Cell culture carrier and method for producing cell culture carrier

Abstract

The present invention pertains to a cell culture carrier containing a halogen and a polymer having a hydroxyl group. The halogen content of the cell culture carrier measured by X-ray photoelectron spectroscopy is 3-60 atom%, and the saturation content of the polymer by phosphate buffered saline (PBS (-)) at 25°C is 10-99 mass%.

Description

Cell culture carrier and method for producing the cell culture carrier

[0001] This patent application claims priority under the Paris Convention to Japanese Patent Application No. 2024-086210 (filing date: May 28, 2024), the entire contents of which are incorporated herein by reference. The present invention relates to a cell culture carrier and a method for producing the cell culture carrier.

[0002] It is known that substrates onto which biologically derived cell adhesion factors such as collagen are immobilized to impart cell adhesion properties are used as cell culture carriers. However, biologically derived cell adhesion factors are sometimes avoided for safety reasons, and demonstrating their safety requires time and cost. A cell culture carrier that does not contain biologically derived cell adhesion factors is known, comprising a substrate and a hydrophilic polymer-containing layer having a thickness of 5 to 2000 nm covering at least a portion of the substrate's surface. The substrate has island-shaped regions (A) having cell adhesive properties and cell proliferation properties, and a region (B) adjacent to the region (A) that does not have cell adhesive properties or cell proliferation properties, and the height of the irregularities at the boundary between the regions (A) and (B) is 1 to 500 nm (Patent Document 1). The substrate in this document is a hard substrate such as polystyrene, and the region (A) on its surface is formed by covering the hydrophilic polymer-containing layer on the substrate with a patterned mask, performing plasma treatment, and modifying the unmasked region. This document does not disclose a cell culture substrate containing a halogen.

[0003] WO2022 / 230734 publication

[0004] New cell culture carriers are constantly being sought. The problem to be solved by the present invention is to provide a cell culture carrier that has cell adhesive properties even without containing a cell adhesion factor of biological origin.

[0005] The present inventors conducted extensive research into cell culture supports to solve the above-mentioned problems, and have now completed the present invention. Specifically, the present invention encompasses the following preferred embodiments. [1] A cell culture support comprising a polymer having a hydroxyl group and a halogen, wherein the halogen content of the cell culture support measured by X-ray photoelectron spectroscopy is 3 to 60 atomic %, and the saturation content of the polymer in phosphate buffered saline (PBS(-)) at 25°C is 10 to 99 mass %. [2] The cell culture support according to [1], wherein the polymer is a hydrogel-forming polymer. [3] The cell culture support according to [2], wherein the hydrogel-forming polymer in an equilibrium swollen state in phosphate buffered saline (PBS(-)) is in the form of particles with an average particle size of 10 to 5,000 μm. [4] The cell culture support according to any one of [1] to [3], wherein the ratio of the OH peak area to the area of ​​the O1s spectrum measured by X-ray photoelectron spectroscopy of the cell culture support is 1 to 100%. [5] The cell culture carrier according to any one of [1] to [4], wherein the halogen is introduced by plasma treatment. [6] The cell culture carrier according to any one of [1] to [5], wherein the halogen is fluorine. [7] The cell culture carrier according to any one of [1] to [6], wherein the polymer has one or more shapes selected from the group consisting of irregular particles, spherical particles, micromolded bodies, arbitrarily shaped articles formed by 3D printing, films, threads, fabrics, hollow fibers, porous monoliths, and coated articles. [8] The cell culture carrier according to any one of [1] to [7], wherein the molar ratio of C═O to C—C of the cell culture carrier measured by X-ray photoelectron spectroscopy is 8 to 90. [9] The cell culture carrier according to any one of [1] to [8], wherein at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells is further present on the surface.

[10] The cell culture carrier according to [9], wherein the cell adhesion factor is a peptide.

[11] A method for producing a cell culture carrier, comprising mixing the cell culture carrier according to any one of [1] to [8] with a solution containing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells, and immobilizing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells on the surface of the cell culture carrier.

[12] A method for producing the cell culture carrier according to

[11] , wherein the cell adhesion factor is a peptide.

[13] A method for producing a cell culture carrier containing a polymer having hydroxyl groups and a halogen, comprising subjecting the polymer having hydroxyl groups to a plasma treatment in the presence of a halogenated hydrocarbon gas, wherein the halogen content of the cell culture carrier is 3 to 60 atomic % as measured by X-ray photoelectron spectroscopy, and the saturation content of the polymer in phosphate buffered saline (PBS(-)) at 25°C is 10 to 99 mass %.

[14] The method according to

[13] , wherein the polymer is a hydrogel-forming polymer.

[15] The method according to

[13] or

[14] , wherein the polymer in a dry state is subjected to a plasma treatment.

[16] The method according to any one of

[13] to

[15] , wherein the plasma treatment is a treatment of irradiating plasma at an irradiation intensity of 50 W or more.

[17] The method according to any one of

[13] to

[16] , wherein the obtained cell culture carrier is mixed with a solution containing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells, to further immobilize at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells on the surface of the cell culture carrier.

[18] The method according to

[17] , wherein the cell adhesion factor is a peptide.

[0006] According to the present invention, it is possible to provide a cell culture carrier that has cell adhesive properties even without containing a cell adhesion factor of biological origin.

[0007] Hereinafter, embodiments of the present invention will be described in detail. Note that the scope of the present invention is not limited to the embodiments described here, and various modifications can be made without departing from the spirit of the present invention.

[0008] [Cell Culture Carrier] The cell culture carrier of the present invention comprises a polymer having a hydroxyl group (hereinafter, may be referred to as a "hydroxyl group-containing polymer") and a halogen, and the halogen content of the cell culture carrier measured by X-ray photoelectron spectroscopy (hereinafter, may be abbreviated as "XPS") is 3 to 60 atomic %, and the saturated content of the polymer in phosphate buffered saline (PBS(-)) at 25°C (hereinafter, may be referred to as "saturated PBS(-) content") is 10 to 99 mass %.

[0009] The inventors unexpectedly discovered that when a hydroxyl-containing polymer with a specific saturated PBS(-) content contains a specific amount of halogen as measured by XPS, it exhibits cell adhesiveness even without the presence of a biological cell adhesion factor. While the reason for this is unclear, the following non-limiting mechanism is hypothesized. XPS is a surface analysis method that detects photoelectrons emitted when a sample surface is irradiated with X-rays to analyze the elemental analysis or chemical bonding state of the sample surface. Therefore, halogens measured by XPS are present on the surface of the sample. Because halogens are hydrophobic, they have difficulty accessing the hydrophilic portions of the hydroxyl-containing polymer, and as a result, tend to be present on the surface of the hydroxyl-containing polymer. For example, when a hydroxyl-containing polymer has a high saturated PBS(-) content or is a hydrogel-forming polymer and the polymer is immersed in a liquid such as water, halogens tend to remain on the surface of the hydroxyl-containing polymer rather than penetrating into the interior of the hydroxyl-containing polymer as it swells with the liquid due to their hydrophobicity. In one embodiment of the present invention, the halogen is introduced by plasma treatment, and the tendency for the halogen to be present on the surface of the hydroxyl-containing polymer results in the region of the hydroxyl-containing polymer that has been modified by plasma treatment to impart cell adhesiveness to the hydroxyl-containing polymer being maintained on the surface of the hydroxyl-containing polymer, thereby enabling the cell culture support to have cell adhesiveness on its surface (even when the hydroxyl-containing polymer has a high saturated PBS(-) content or is a hydrogel-forming polymer). In another embodiment of the present invention, the cell culture support contains a cell adhesion factor, and when the cell adhesion factor is immobilized on a cell culture support having a halogen on its surface, a hydrophobic interaction occurs between the halogen and the cell adhesion factor, thereby enabling the cell culture support to have cell adhesiveness.In another embodiment of the present invention, the cell culture carrier contains a cell-interacting protein and / or a cell-interacting peptide (hereinafter, the cell-interacting protein and the cell-interacting peptide may be collectively referred to as "cell-interacting protein"). When the cell-interacting protein is immobilized on a cell culture carrier having a halogen on its surface, the halogen and the cell-interacting protein exhibit hydrophobic interactions, thereby providing the cell culture carrier with functionality such as differentiation induction. Because the cell culture carrier of the present invention has cell adhesive properties even without containing a biologically derived cell adhesion factor, users can use the cell culture carrier of the present invention without worrying about the safety associated with biologically derived cell adhesion factors. Furthermore, since any cell adhesion factor can be immobilized on the surface of the cell culture carrier of the present invention, users can immobilize any cell adhesion factor to the cell culture carrier of the present invention as desired. Furthermore, any cell-interacting protein can be immobilized on the cell culture carrier of the present invention in addition to or instead of a cell adhesion factor.

[0010] <Halogen> From the viewpoint of cell adhesiveness and / or immobilization of cell adhesion factors or cell-interacting proteins, the halogen content of the cell culture carrier measured by XPS is 3 to 60 atomic %, preferably 3.5 to 59 atomic %, and more preferably 4 to 58 atomic % (e.g., 8 to 57 atomic %, 10 to 57 atomic %, 15 to 50 atomic %, or 20 to 45 atomic %). A halogen content equal to or greater than the lower limit indicates that a sufficient amount of halogen is present on the surface of the cell culture carrier. Therefore, the cell culture carrier can have superior cell adhesiveness even without containing a biologically derived cell adhesion factor, and can also have superior immobilization ability for cell adhesion factors or cell-interacting proteins. This can also be achieved, for example, when the hydroxyl group-containing polymer has a high saturated PBS(-) content or is a hydrogel-forming polymer. The halogen content can be measured by the method described in the Examples below. The halogen content can be adjusted within the above range by adjusting the plasma treatment conditions when the halogen is introduced by plasma treatment, or by adjusting the reaction conditions of chemical modification when the halogen is introduced by chemical modification.

[0011] In one embodiment of the present invention, the halogen is preferably introduced by plasma treatment. In this embodiment, the plasma treatment alone can modify the surface of the hydroxyl-containing polymer to impart cell adhesiveness, and also can introduce the halogen into the hydroxyl-containing polymer. Alternatively, the halogen may be introduced into the hydroxyl-containing polymer by chemical modification.

[0012] The halogen contained in the cell culture carrier is preferably fluorine, from the viewpoint of more favorable cell adhesiveness. Fluorine tends to be more present on the surface of hydroxyl-containing polymers. For example, when the hydroxyl-containing polymer has a high saturated PBS(-) content or is a hydrogel-forming hydroxyl-containing polymer, fluorine tends to be retained on the surface of the hydroxyl-containing polymer even when the hydroxyl-containing polymer is impregnated with a liquid such as water. Therefore, fluorine-containing cell culture carriers can have more favorable cell adhesiveness. Here, a hydrogel-forming polymer or a hydrogel-forming hydroxyl-containing polymer refers to a polymer or a hydroxyl-containing polymer, respectively, that has a crosslinked and / or network structure and has the property of being able to form a hydrogel by retaining a liquid such as water (within the structure). A hydrogel refers to a gel containing at least a polymer (polymer) having a crosslinked and / or network structure and a liquid such as water retained within the structure.

[0013] The molar ratio of C═O to C—C of the cell culture carrier, as measured by X-ray photoelectron spectroscopy, is preferably 8 to 90, more preferably 10 to 85, even more preferably 13 to 85 (e.g., 15 to 80 or 18 to 75), and particularly preferably 20 to 85 (e.g., 20 to 80 or 20 to 70). When the molar ratio is within the above range, the cell culture carrier can have more favorable cell adhesiveness. The molar ratio can be adjusted within the above range by adjusting the plasma irradiation conditions when halogen is introduced by plasma treatment, or by adjusting the reaction conditions of chemical modification when halogen is introduced by chemical modification. The molar ratio in the hydroxyl group-containing polymer can be measured, for example, by the method described in the Examples below.

[0014] The ratio of the OH peak area (derived from OH) in the O1s spectrum to the area of ​​the O1s spectrum (the sum of all peak areas in the O1s spectrum) measured by X-ray photoelectron spectroscopy of the cell culture carrier is preferably 1 to 100%, more preferably 30 to 100%, even more preferably 50 to 100%, particularly preferably 60 to 100% (e.g., 60 to 95%), and even more particularly preferably 70 to 100% (e.g., 70 to 90%). When the hydroxyl-containing polymer is a hydroxyl-containing polymer or a hydrogel-forming hydroxyl-containing polymer having a high saturated PBS(-) content and contains a relatively large amount of hydroxyl groups (e.g., 30 mol% or more), when the hydroxyl-containing polymer is swollen with a liquid such as water, a buffer solution, or a culture medium, regions that are preferably present on the surface of the hydroxyl-containing polymer (e.g., regions that have been imparted with cell adhesive properties) may penetrate into the interior together with the hydroxyl groups present on the surface. However, because the cell culture carrier of the present invention contains a halogen, even in the above-mentioned cases, the above-mentioned preferred regions can be reduced from penetrating into the interior of the swollen body. Furthermore, when the hydroxyl-containing polymer is a hydroxyl-containing polymer with a high saturated PBS(-) content or a hydrogel-forming hydroxyl-containing polymer and contains a relatively small amount of hydroxyl groups (e.g., less than 10 mol%), when the hydroxyl-containing polymer is swollen with the above-mentioned liquid, the low amount of hydroxyl groups reduces the tendency for the preferred regions present on the surface of the hydroxyl-containing polymer to penetrate into the interior. Therefore, even in the above-mentioned cases, the above-mentioned preferred regions can be suitably maintained on the surface of the hydroxyl-containing polymer. Thus, whether the amount of hydroxyl groups is high or low, the cell culture carrier can exhibit suitable cell adhesiveness. When the hydroxyl-containing polymer is a crosslinked vinyl alcohol polymer, the amount of hydroxyl groups contained in the hydroxyl-containing polymer can be adjusted within the above-mentioned range by adjusting the degree of saponification. When the hydroxyl group-containing polymer is an ethylene-vinyl alcohol copolymer, the amount of hydroxyl groups contained in the hydroxyl group-containing polymer can be adjusted within the above range by adjusting the amounts of ethylene and vinyl ester as raw materials and / or the degree of saponification.

[0015] <Hydroxyl Group-Containing Polymer> The cell culture carrier of the present invention comprises one or more hydroxyl group-containing polymers. The saturated PBS(-) content of the hydroxyl group-containing polymer, i.e., the PBS(-) content in the hydroxyl group-containing polymer when saturated with PBS(-) at 25°C, is 10 to 99% by mass, preferably 15 to 97% by mass, more preferably 20 to 95% by mass, and particularly preferably 25 to 90% by mass. Hydroxyl group-containing polymers with a higher saturated PBS(-) content swell more significantly upon incorporation of the liquid during use, etc., but can be stored and transported with a reduced liquid content, providing significant advantages in terms of volume and weight during storage and transport compared to cell culture carriers containing hard substrates. Furthermore, even a small amount of cell culture carrier can provide a larger specific surface area after swelling, i.e., a sufficient surface area of ​​the cell culture carrier useful for cell culture can be secured. Therefore, a major advantage of the present invention is that it is possible to impart cell adhesiveness or the ability to immobilize cell adhesion factors or cell-interacting proteins, even to hydroxyl-containing polymers with a higher saturated PBS(-) content. The saturated PBS(-) content of the hydroxyl-containing polymer at 25°C can be adjusted within the above range, for example, by adjusting the proportion of hydroxyl groups in the hydroxyl-containing polymer and / or the type of functional group contained in the hydroxyl-containing polymer. The saturated PBS(-) content of the hydroxyl-containing polymer at 25°C can be measured by the method described in the Examples below.

[0016] In one embodiment of the present invention, the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer. When the cell culture carrier of the present invention contains a hydrogel-forming hydroxyl-containing polymer, it swells upon incorporation of the liquid during use to form a hydrogel. Because the cell culture carrier can be stored and transported with a reduced liquid content, the cell culture carrier offers significant advantages in terms of volume and weight during storage and transport compared to cell culture carriers containing a hard substrate. Furthermore, even a small amount of cell culture carrier can provide a larger specific surface area after swelling, i.e., a more sufficient surface area of ​​the cell culture carrier useful for cell culture. Furthermore, compared to cell culture carriers containing a hard substrate, the cell culture carrier is less susceptible to defects such as cracks or chips, allowing users to receive cell culture carriers with fewer impurities. Preferred examples of the hydrogel-forming hydroxyl-containing polymer include crosslinked vinyl alcohol polymers, modified vinyl alcohol polymers, polysaccharides, and combinations thereof.

[0017] In another embodiment of the present invention, the hydroxyl-containing polymer is a hydroxyl-containing polymer that is not a hydrogel-forming hydroxyl-containing polymer. Preferred examples of hydroxyl-containing polymers that are not hydrogel-forming hydroxyl-containing polymers include ethylene-vinyl alcohol copolymers.

[0018] In a preferred embodiment, the hydroxyl group-containing polymer is one or more crosslinked vinyl alcohol polymers or one or more ethylene-vinyl alcohol copolymers.

[0019] When the hydroxyl-containing polymer is a combination of two or more hydroxyl-containing polymers, it is preferred that at least one hydroxyl-containing polymer, preferably more than one hydroxyl-containing polymer, and more preferably all of the hydroxyl-containing polymers have the desired properties for a hydroxyl-containing polymer (e.g., saturated PBS(-) content at 25°C, amount of hydroxyl groups, molar ratio of C=O to C-C, weight average molecular weight, etc.).

[0020] In one embodiment, the hydroxyl group-containing polymer has one or more shapes selected from the group consisting of particles (e.g., irregular particles or spherical particles), micromolded bodies, arbitrarily shaped articles formed by a 3D printer, films, threads, fabrics, hollow fibers, porous monoliths, and coated articles.

[0021] Spherical particles include not only perfectly spherical particles, but also oval spherical particles, spherical particles with missing or protruding portions, oval spherical particles with missing or protruding portions, spherical particles with protrusions or wrinkles on the surface, and porous spheres. A micromolded body is a molded body having fine irregularities on its surface or inside. When the hydroxyl group-containing polymer is a hydrogel-forming hydroxyl group-containing polymer, the size of the microfabrication in the equilibrium swelling state is typically 10 to 1000 μm. When the hydroxyl group-containing polymer is a hydroxyl group-containing polymer other than a hydrogel-forming hydroxyl group-containing polymer, the size of the microfabrication is typically 10 to 1000 μm. An arbitrary shape molded using a 3D printer refers to any shape that can be molded using, for example, a stereolithography, inkjet, or nozzle extrusion 3D printer. Throughout this specification, the equilibrium swelling state refers to a state in which the material is swollen with liquid until equilibrium is reached.

[0022] In a preferred embodiment when the hydroxyl group-containing polymer is a hydrogel-forming hydroxyl group-containing polymer, the hydroxyl group-containing polymer in an equilibrium swollen state in phosphate buffered saline (PBS(-)) is in the form of particles having an average particle size of preferably 10 to 5,000 μm, more preferably 20 to 4,500 μm, even more preferably 30 to 4,000 μm, still more preferably 30 to 3,000 μm, still more preferably 30 to 2,000 μm, and particularly preferably 30 to 1,500 μm.

[0023] When the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, its average particle size can be measured using a laser diffraction / scattering particle size distribution analyzer, as described in the Examples below. The average particle size in the present invention is the volume-based average particle size of the particle diameter (sphere-equivalent diameter) in an equilibrium swollen state. Therefore, even when measuring using a microscope, the average particle size can be measured by swelling and observing the particles as described in the Examples below. Furthermore, when the hydroxyl-containing polymer is swollen in a liquid other than PBS(-) (e.g., a liquid medium), the hydroxyl-containing polymer can be washed with water and dried using a conventional method, and then brought to an equilibrium swollen state in PBS(-), thereby measuring the average particle size of the hydroxyl-containing polymer in an equilibrium swollen state in PBS(-). An average particle size within the above range is preferable because it provides excellent handleability and allows for easy and rapid separation of the detached cells from the cell culture carriers in the separation step described below. The easy and rapid separation not only improves cell production efficiency but also better maintains the activity of the produced cells, thereby achieving more stable cell production. This advantage is also useful when the culture vessel is a large-capacity bioreactor. The average particle size can be adjusted to within the above range, for example, by adopting an appropriate molding method in the process of preparing the hydroxyl group-containing polymer and / or selecting a classification method after molding.

[0024] In a preferred embodiment when the hydroxyl-containing polymer is a hydroxyl-containing polymer other than a hydrogel-forming hydroxyl-containing polymer, the hydroxyl-containing polymer is in the form of particles having an average particle size of preferably 10 to 5,000 μm, more preferably 20 to 4,500 μm, even more preferably 30 to 4,000 μm, even more preferably 30 to 3,000 μm, even more preferably 30 to 2,000 μm, and particularly preferably 30 to 1,500 μm. The average particle size can be measured using a laser diffraction / scattering particle size distribution analyzer or by observation with a microscope. The average particle size can be adjusted to within the above range, for example, by employing an appropriate molding method in the process of preparing the hydroxyl-containing polymer and / or selecting a classification method after molding.

[0025] The hydroxyl group-containing polymer is preferably in the form of particles, more preferably spherical particles, but is also preferably in the form of a film, thread, fabric or coated article.

[0026] In one embodiment in which the hydroxyl-containing polymer is in the form of a film, for example, when the film-like hydroxyl-containing polymer is used in a fluidized-bed bioreactor or stirred-bed bioreactor, as described below, the film-like hydroxyl-containing polymer must be dispersed in the composition. Therefore, when the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length of the film-like hydroxyl-containing polymer in an equilibrium swollen state in a liquid medium (particularly the liquid medium contained in the composition in the culture vessel) are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm. Furthermore, when the hydroxyl-containing polymer is a hydroxyl-containing polymer other than a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length of the film-like hydroxyl-containing polymer are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm. On the other hand, when the film-shaped hydroxyl group-containing polymer is used in a fixed-bed bioreactor as described later, this does not apply, and a larger film-shaped hydroxyl group-containing polymer may be used, for example, in the form of a spiral membrane. In this case, from the viewpoint of maintaining the mechanical strength of the film-shaped hydroxyl group-containing polymer, if the hydroxyl group-containing polymer is a hydrogel-forming hydroxyl group-containing polymer, the thickness of the film-shaped hydroxyl group-containing polymer is preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm; the width is preferably 10 to 5,000 mm, more preferably 20 to 3,000 mm, and even more preferably 30 to 2,000 mm; and the length is preferably 0.5 to 100 m, more preferably 1 to 50 m, more preferably 2 to 30 m, and when the hydroxyl group-containing polymer is a hydroxyl group-containing polymer other than a hydrogel-forming hydroxyl group-containing polymer, the thickness is preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm, the width is preferably 10 to 5,000 mm, more preferably 100 to 3,000 mm, and even more preferably 300 to 2,000 mm, and the length is preferably 0.5 to 100 m, more preferably 1 to 50 m, and even more preferably 2 to 30 m.When the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length are the dimensions of the film-like hydroxyl-containing polymer brought to an equilibrium swollen state in a liquid medium (particularly the liquid medium contained in the composition in the culture vessel). The thickness, width, and length of the film-like hydroxyl-containing polymer can be measured by observing the film-like hydroxyl-containing polymer under a microscope or the like.

[0027] In one embodiment in which the hydroxyl-containing polymer is filamentous, for example, when the filamentous hydroxyl-containing polymer is used in a fluidized-bed bioreactor or stirred-bed bioreactor, as described below, the filamentous hydroxyl-containing polymer must be dispersed in the composition. Therefore, when the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, the diameter and length of the filamentous hydroxyl-containing polymer in an equilibrium swollen state in a liquid medium (particularly a liquid medium contained in a composition in a culture vessel) are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm. When the hydroxyl-containing polymer is a hydroxyl-containing polymer other than a hydrogel-forming hydroxyl-containing polymer, the diameter and length of the filamentous hydroxyl-containing polymer are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm. The diameter and length of the hydroxyl group-containing polymer threads can be measured by observing the hydroxyl group-containing polymer threads under a microscope or the like.

[0028] The fabric of the present invention includes, for example, woven fabrics, knitted fabrics, and nonwoven fabrics. In one embodiment in which the hydroxyl-containing polymer is in the form of a fabric, for example, when the fabric-like hydroxyl-containing polymer is used in a fluidized-bed bioreactor or stirred-bed bioreactor described below, the fabric-like hydroxyl-containing polymer must be dispersed in the composition. Therefore, when the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length of the fabric-like hydroxyl-containing polymer brought to an equilibrium swollen state in a liquid medium (particularly the liquid medium contained in the composition in the culture vessel) are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm, respectively. When the hydroxyl-containing polymer is a hydroxyl-containing polymer other than a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length of the cloth-like hydroxyl-containing polymer are preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm. However, when the cloth-like hydroxyl-containing polymer is used in a fixed-bed bioreactor, as described below, this does not apply, and a larger cloth-like hydroxyl-containing polymer may be used, for example, in the form of a spiral membrane. In this case, when the hydroxyl group-containing cloth polymer is a hydrogel-forming hydroxyl group-containing polymer, from the viewpoint of maintaining the mechanical strength of the hydroxyl group-containing cloth polymer, the thickness is preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm; the width is preferably 10 to 5,000 mm, more preferably 20 to 3,000 mm, and even more preferably 30 to 2,000 mm; the length is preferably 0.5 to 100 m, more preferably 1 to 50 m; When the hydroxyl group-containing polymer is a hydroxyl group-containing polymer other than a hydrogel-forming hydroxyl group-containing polymer, the thickness is preferably 10 to 5,000 μm, more preferably 30 to 2,000 μm, and even more preferably 30 to 1,000 μm, the width is preferably 10 to 5,000 mm, more preferably 100 to 3,000 mm, and even more preferably 300 to 2,000 mm, and the length is preferably 0.5 to 100 m, more preferably 1 to 50 m, and even more preferably 2 to 30 m.When the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, the thickness, width, and length are the dimensions of the cloth-like hydroxyl-containing polymer in an equilibrium swollen state in a liquid medium (particularly the liquid medium contained in the composition in the culture vessel). The thickness, width, and length of the cloth-like hydroxyl-containing polymer can be measured by observing the cloth-like hydroxyl-containing polymer under a microscope or the like.

[0029] The coated article refers to a substrate (e.g., the amorphous particles, spherical particles, micromolded bodies, arbitrarily shaped articles formed with a 3D printer, films, trays, threads, cloths, hollow fibers, porous monoliths, plates, well plates, flasks (including T-flasks, shaker flasks, spinner flasks, etc.), chamber slides, dishes, tubes, bottles, roller bottles, bags (including shaker bags)) typically used as culture vessels, which are described below, coated with a hydroxyl group-containing polymer. The substrate material can be freely selected from one or a combination of two or more of glass, polyolefin, polymethyl methacrylate, polystyrene, polyester, polyolefin, ethylene-vinyl alcohol copolymer, polyamide, polyimide, etc. When the hydroxyl-containing polymer is a hydrogel-forming hydroxyl-containing polymer, from the viewpoint of preventing peeling from the substrate when the coated hydroxyl-containing polymer swells in a liquid medium, the thickness of the hydroxyl-containing polymer layer in the coated article brought to an equilibrium swollen state in a liquid medium (particularly a liquid medium contained in a composition in a culture vessel) is preferably 0.05 to 1,000 μm, more preferably 0.1 to 700 μm, and even more preferably 0.2 to 500 μm. The thickness of the hydroxyl-containing polymer layer can be measured by a method in which a cross section of the hydroxyl-containing polymer layer is photographed with an electron microscope after drying the coated article and the thickness of the hydroxyl-containing polymer layer measured with an electron microscope is converted to the thickness in an equilibrium swollen state, a method in which a portion of the hydroxyl-containing polymer layer is peeled off and measured with a film thickness step meter, or a method using an ellipsometer or the like.

[0030] The content of the hydroxyl group-containing polymer in the cell culture carrier is preferably 50% by mass or more to less than 100% by mass, more preferably 60% to less than 100% by mass, even more preferably 70% to less than 100% by mass, and particularly preferably 80% to less than 100% by mass (e.g., 90% to less than 100% by mass or 95% to less than 100% by mass), based on the total mass of the cell culture carrier in a dry state. Throughout this specification, "dry state" refers to a state in which volatile components such as water have been reduced. For example, the substance (e.g., hydrogel) can be dried by drying such as hot air drying, aeration drying, vacuum drying, or freeze drying until the mass of the substance becomes constant. When the cell culture carrier contains a cell adhesion factor or a cell-interacting protein, etc., as described below, the proportion of the hydroxyl group-containing polymer in the cell culture carrier can be determined, for example, by determining the proportion of the cell adhesion factor or the cell-interacting protein, etc., by, for example, the ninhydrin method, the Lowry method, the BCA method, the ELISA method, or a radiolabeling method, and then subtracting the proportion of the cell adhesion factor or the cell-interacting protein, etc., from the total mass of the cell culture carrier.

[0031] <Crosslinked vinyl alcohol polymer> The crosslinked vinyl alcohol polymer that can be used in the present invention is not particularly limited as long as it contains more than 50 mol% of vinyl alcohol-derived structural units relative to the total structural units constituting the vinyl alcohol polymer, and may also contain structural units derived from vinyl esters. The total amount of vinyl alcohol-derived structural units and vinyl ester-derived structural units relative to the total structural units constituting the vinyl alcohol polymer is preferably 80 mol% or more, more preferably 90 mol% or more, and even more preferably 95 mol% or more. When the cell culture carrier contains a crosslinked vinyl alcohol polymer, the cell culture carrier may contain one crosslinked vinyl alcohol polymer, or two or more crosslinked vinyl alcohol polymers that differ in one or more of the hydroxyl group amount, the viscosity-average degree of polymerization (described below), the degree of saponification, and the 4% viscosity. When the cell culture carrier contains two or more crosslinked vinyl alcohol polymers, it is preferable that at least one of the vinyl alcohol polymers has a viscosity-average degree of polymerization, a degree of saponification, and / or a 4% by mass viscosity within a preferred range, and it is more preferable that all of the vinyl alcohol polymers have a viscosity-average degree of polymerization, a degree of saponification, and / or a 4% by mass viscosity within a preferred range.

[0032] The viscosity-average degree of polymerization of the vinyl alcohol polymer measured in accordance with JIS K 6726:1994 is preferably 300 or more, more preferably 450 or more, even more preferably 500 or more, and may be 1,000 or more or 1,500 or more, from the viewpoint of suppressing embrittlement when a crosslinked vinyl alcohol polymer comes into contact with a liquid and swells. Furthermore, from the viewpoint of suppressing an increase in the viscosity of an aqueous solution when preparing a crosslinked vinyl alcohol polymer and improving ease of processability, the viscosity-average degree of polymerization is preferably 10,000 or less, more preferably 5,000 or less, even more preferably 3,500 or less, and still more preferably 2,500 or less.

[0033] The degree of saponification of the vinyl alcohol polymer measured in accordance with JIS K 6726: 1994 is preferably 50 mol% or more, more preferably 60 mol% or more, and even more preferably 65 mol% or more, from the viewpoint of improving the water solubility of the vinyl alcohol polymer. The upper limit of the degree of saponification is not particularly limited, and is, for example, 100 mol%, preferably 99 mol%.

[0034] The 4% by mass viscosity of the vinyl alcohol polymer at 20°C is preferably 0.5 to 110 mPa·s, more preferably 1 to 80 mPa·s, and even more preferably 2 to 60 mPa·s. When the viscosity is within this range, the ease of preparation of the cell culture carrier is improved, and the mechanical strength of the cell culture carrier can be improved. The viscosity is measured at 20°C using a B-type viscometer (rotation speed: 12 rpm) in accordance with the rotational viscometer method of JIS K 6726:1994 for a 4% by mass aqueous solution of the vinyl alcohol polymer.

[0035] The crosslinked vinyl alcohol polymer may be one in which the vinyl alcohol polymer is physically crosslinked or one in which the vinyl alcohol polymer is crosslinked by a covalent bond. Crosslinking by a covalent bond is preferred because it increases the stability of the crosslinked vinyl alcohol polymer when swollen with a liquid. Examples of physical crosslinking methods and covalent crosslinking methods are described in the section "Method for producing a cell culture carrier" below. Furthermore, as described in the same section, the crosslinked vinyl alcohol polymer may be a crosslinked vinyl alcohol polymer that has been modified.

[0036] <Polysaccharides> The polysaccharides are not particularly limited, and examples thereof include starch, cellulose, chitin, dextran, agarose, heparin, alginic acid, hyaluronic acid, glucomannan, and combinations of two or more thereof.

[0037] <Ethylene-vinyl alcohol copolymer> The ethylene-vinyl alcohol copolymer that can be contained in the cell culture carrier may be a resin obtained by saponifying a copolymer of ethylene and a vinyl ester by a known method. When the cell culture carrier contains an ethylene-vinyl alcohol copolymer, the cell culture carrier may contain one ethylene-vinyl alcohol copolymer, or two or more ethylene-vinyl alcohol copolymers that differ from each other in one or more of the weight-average molecular weight, the amount of hydroxyl groups, the ethylene content, the degree of saponification, etc.

[0038] The ethylene content of the ethylene-vinyl alcohol copolymer is preferably 20 to 60 mol%, more preferably 25 to 55 mol%, even more preferably 25 to 50 mol%, and particularly preferably 27 to 48 mol%, based on a repeating unit consisting of two carbon atoms in the main chain of the ethylene-vinyl ester copolymer, which is the raw material for producing the ethylene-vinyl alcohol copolymer (e.g., a vinyl ester unit, an ethylene unit, etc.). When the ethylene content is within the above range, the strength tends to be excellent. The ethylene content of the ethylene-vinyl alcohol copolymer can be adjusted to within the above range by adjusting the ratio of ethylene to other monomers when preparing the ethylene-vinyl ester copolymer, which is the raw material for producing the ethylene-vinyl alcohol copolymer.

[0039] The ethylene-vinyl alcohol copolymer may contain structural units other than ethylene units, vinyl alcohol units, and vinyl ester units, as long as the object of the present invention is not impaired. When the ethylene-vinyl alcohol copolymer contains the other structural units, the content of the other structural units relative to the total structural units of the ethylene-vinyl alcohol copolymer is preferably 30 mol% or less, more preferably 20 mol% or less, even more preferably 10 mol% or less, still more preferably 5 mol% or less, and particularly preferably 1 mol% or less. Furthermore, when the ethylene-vinyl alcohol copolymer contains the other structural units, the content thereof may be 0.05 mol% or more, or may be 0.10 mol% or more. Examples of the other structural units include structural units derived from unsaturated carboxylic acids such as acrylic acid, methacrylic acid, crotonic acid, itaconic acid, or the like, or anhydrides, salts, or mono- or dialkyl esters thereof; nitriles such as acrylonitrile, methacrylonitrile, or the like; amides such as acrylamide, methacrylamide, or the like; olefin sulfonic acids such as vinyl sulfonic acid, allyl sulfonic acid, methallylsulfonic acid, or the like, or salts thereof; vinyl silane compounds such as vinyltrimethoxysilane, vinyltriethoxysilane, vinyltri(β-methoxyethoxy)silane, γ-methacryloxypropylmethoxysilane, or the like; alkyl vinyl ethers, vinyl ketone, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, or the like.

[0040] The vinyl alcohol portion of the ethylene-vinyl alcohol copolymer may be modified with other functional groups, provided that the physical properties of the cell culture carrier are not significantly impaired. This can improve the ability of the cell culture carrier to immobilize cell adhesion factors, cell-interacting proteins, and the like. Examples of such functional groups include hydroxyl groups, epoxy groups, amino groups, and isocyanate groups. When these functional groups are used to modify the side chains, the amount of the functional groups is typically 1 to 20 mol %, preferably 3 to 15 mol %, and more preferably 5 to 10 mol %, in terms of the number of functional groups relative to the number of vinyl ester monomer units.

[0041] The weight-average molecular weight of the ethylene-vinyl alcohol copolymer is preferably 10,000 to 60,000, more preferably 15,000 to 55,000, and particularly preferably 20,000 to 50,000. When the weight-average molecular weight of the ethylene-vinyl alcohol copolymer is within this range, it tends to have excellent moldability. The weight-average molecular weight of the ethylene-vinyl alcohol copolymer can be adjusted to be within the above range by adjusting the weight-average molecular weight of the ethylene-vinyl ester copolymer, which is a raw material for the ethylene-vinyl alcohol copolymer, or by combining ethylene-vinyl ester copolymers with different weight-average molecular weights to prepare the ethylene-vinyl alcohol copolymer.

[0042] From the viewpoint of thermal stability and / or moisture resistance, the saponification degree of the ethylene-vinyl alcohol copolymer is preferably 90 mol% or more, more preferably 95 mol% or more, and particularly preferably 99 mol% or more. The upper limit of the saponification degree is not limited, and may be 100 mol% or less, 99.97 mol% or less, or 99.94 mol% or less.

[0043] <Cell Adhesion Factor> In one embodiment of the present invention, the cell culture carrier may contain a cell adhesion factor. In this case, the cell culture carrier may contain the cell adhesion factor in a state in which the cell adhesion factor is simply contained therein, in a state in which the cell adhesion factor is bound to the cell culture carrier via covalent bonds and / or hydrophobic interactions, or in a combination thereof. From the viewpoint of ensuring stable function of the cell adhesion factor, it is preferable that the cell adhesion factor is contained in the cell culture carrier in a state in which the cell adhesion factor is bound to the cell culture carrier via covalent bonds and / or hydrophobic interactions (i.e., forming a complex). According to the studies of the present inventors, it has been found that the cell adhesion factor is highly maintained in the cell culture carrier. This is thought to be because, for at least some of the cell adhesion factors, the cell culture carrier of the present invention contains a halogen, which causes the halogen and the cell adhesion factor to bond via hydrophobic interactions to form a complex, and / or because C═O, which is usually or preferably contained in the hydroxyl group-containing polymer in the cell culture carrier of the present invention, forms a covalent bond with the cell adhesion factor, thereby forming a complex between the cell culture carrier and the cell adhesion factor.

[0044] The cell adhesion factor is not particularly limited as long as it is a molecule that has the property of adhering cells. Examples thereof include cell adhesion proteins such as gelatin, collagen, laminin, fibronectin, vitronectin, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, fibrin, elastin, netrin, entactin, proteoglycan, and retronectin; synthetic peptides containing cell adhesive sequences such as RGD, RGDVF, REDV, YIGSR, and IKVAV; and Synthemax (registered trademark) Examples of the cell adhesion factor include hydrophilic polymers containing peptides such as PEG-10 PEG-11 PEG-12 PEG-13 PEG-14 PEG-15 PEG-16 PEG-17 PEG-18 PEG-19 PEG-20 PEG-21 PEG-22 PEG-23 PEG-24 PEG-25 PEG-26 PEG-27 PEG-28 PEG-29 PEG-30 PEG-31 PEG-32 PEG-33 PEG-34 PEG-35 PEG-36 PEG-37 PEG-38 PEG-39 PEG-40 PEG-41 PEG-42 PEG-43 PEG-44 PEG-45 PEG-46 PEG-47 PEG-48 PEG-49 PEG-49 PEG-49 PEG-48 PEG-49 PEG-49 PEG-41 PEG-42 PEG-44 PEG-45 PEG-46 PEG-47 PEG-48 PEG-49 ...3 PEG-44 PEG-45 PEG-45 PEG-46 PEG-47 PEG-48 PEG-49 PEG-48 PEG-49 PEG-49 PEG-49 PEG-41 PEG-41 PEG-42 PEG-43 PEG-44 PEG-45 PEG-45 PEG-46 PEG-47 PEG-48 PEG-49 PEG-48 PEG-49 PEG-49 PEG-49 PEG-4

[0045] The cell adhesion protein may be naturally occurring, recombinant, or a synthetic peptide chemically synthesized by solid-phase methods or the like. From the viewpoint of eliminating safety concerns, cell adhesion factors that are not derived from living organisms are preferred. When a cell adhesion protein is used as the cell adhesion factor, the molecular weight of the cell adhesion protein is not particularly limited, but a weight-average molecular weight of 5,000 to 1,000,000 is preferred, and a weight-average molecular weight of 6,000 to 800,000 is more preferred. When a synthetic peptide is used as the cell adhesion factor, the molecular weight of the synthetic peptide is not particularly limited, but a weight-average molecular weight of 200 to 10,000 is preferred, and a weight-average molecular weight of 250 to 7,000 is more preferred. When an acidic polysaccharide is used as the cell adhesion factor, the molecular weight of the acidic polysaccharide is not particularly limited, but a weight-average molecular weight of 3,000 to 2,000,000 is preferred, and a weight-average molecular weight of 4,000 to 1,200,000 is more preferred. When polycations are used as cell adhesion factors, the molecular weight of the polycations is not particularly limited, but a weight-average molecular weight of 2,000 to 1,000,000 is preferred, and a weight-average molecular weight of 4,000 to 500,000 is more preferred. When cell growth factors or differentiation factors are used as cell adhesion factors, the molecular weight of the cell growth factors or differentiation factors is not particularly limited, but a weight-average molecular weight of 3,000 to 200,000 is preferred, and a weight-average molecular weight of 5,000 to 100,000 is more preferred. When cell culture carriers contain cell adhesion factors, the cell adhesion factors may be contained alone or in combination of two or more types. Commercially available cell adhesion factors can also be used, such as iMatrix-511 (manufactured by MATRIXOME).

[0046] When the cell culture carrier contains a cell adhesion factor, the proportion of the cell adhesion factor in the cell culture carrier is, for example, 0.00001 to 70% by mass, preferably 0.0001 to 60% by mass, and more preferably 0.0005 to 50% by mass, based on the total mass of the cell culture carrier. The masses of the cell culture carrier and the cell adhesion factor in this case are masses in a dry state.

[0047] <Cell-Interacting Protein, etc.> In one embodiment of the present invention, the cell culture carrier may contain a cell-interacting protein, etc. In this case, the cell culture carrier may contain the cell-interacting protein in a state where it simply contains the cell-interacting protein, in a state where the cell-interacting protein is bound to the cell culture carrier via covalent bonds and / or hydrophobic interactions, or in a combination thereof. From the viewpoint of ensuring stable function of the cell-interacting protein, etc., it is preferable that the cell-interacting protein, etc. is contained in the cell culture carrier in a state where it is bound to the cell culture carrier via covalent bonds and / or hydrophobic interactions (i.e., forming a complex). The inventors' studies have found that the cell-interacting protein, etc. is highly maintained in the cell culture carrier. This is thought to be because, for at least some of the cell-interacting proteins, etc., the cell culture carrier of the present invention contains a halogen, and the halogen and the cell-interacting protein, etc. are bound to each other via hydrophobic interactions, forming a complex between the cell culture carrier and the cell-interacting protein, etc., and / or because C═O, which is usually or preferably contained in the hydroxyl group-containing polymer in the cell culture carrier of the present invention, forms a covalent bond with the cell-interacting protein, etc., forming a complex between the cell culture carrier and the cell-interacting protein, etc.

[0048] The cell-interacting protein is not particularly limited as long as it is a protein or peptide that has the property of interacting with cells. Examples include antibody-recognizing proteins such as protein A, or antibodies themselves.

[0049] When a cell culture carrier contains a cell-interacting protein or the like, the cell-interacting protein or the like may be contained in the cell culture carrier alone or in a combination of two or more types. When a cell culture carrier contains a cell-interacting protein or the like, the proportion of the cell-interacting protein or the like in the cell culture carrier is, for example, 0.00001 to 70% by mass, preferably 0.0001 to 60% by mass, and more preferably 0.0005 to 50% by mass, based on the total mass of the cell culture carrier. In this case, the masses of the cell culture carrier and the cell-interacting protein or the like are masses in a dry state. The cell culture carrier may also contain both a cell adhesion factor and a cell-interacting protein or the like. In such a case, the total proportion of the cell adhesion factor and the cell-interacting protein or the like in the cell culture carrier is, for example, 0.00001 to 70% by mass, preferably 0.0001 to 60% by mass, and more preferably 0.0005 to 50% by mass, based on the total mass of the cell culture carrier.

[0050] <Optional Components Other Than Cell Adhesion Factors and Cell-Interacting Proteins, etc.> Examples of optional components other than cell adhesion factors and cell-interacting proteins, etc., that may be optionally contained in the cell culture carrier include polymer microparticles and inorganic microparticles. These optional components may be contained alone or in combination of two or more. The proportion of the optional components in the cell culture carrier can be selected appropriately. When the cell culture carrier contains one or more selected from the group consisting of polymer microparticles and inorganic microparticles, the proportion is preferably 0.1 to 20 parts by mass, more preferably 0.5 to 20 parts by mass, and even more preferably 1 to 15 parts by mass, per 100 parts by mass of the hydroxyl group-containing polymer, from the viewpoint of improving the mechanical strength of the cell culture carrier. Note that the masses of the hydroxyl group-containing polymer and the optional components in this case are masses in a dry state.

[0051] [Method for producing cell culture carriers] The cell culture carriers of the present invention can be produced, for example, by a method comprising subjecting a polymer having hydroxyl groups to plasma treatment in the presence of a halogenated hydrocarbon gas. Thus, the halogen content of the cell culture carriers produced by this method, as measured by X-ray photoelectron spectroscopy, is 3 to 20 atomic %, and the saturated PBS(-) content of the polymer at 25°C is 10 to 99 mass %. Furthermore, one embodiment or preferred embodiment of the cell culture carrier produced by the above method corresponds to one embodiment or preferred embodiment described in the previous section [Cell Culture Carrier].

[0052] The hydroxyl group-containing polymers described above can be used as the polymer having hydroxyl groups. From the viewpoint of reducing the volume and weight during storage and transportation and providing users with cell culture supports containing few impurities, the hydroxyl group-containing polymer is preferably a hydrogel-forming hydroxyl group-containing polymer, and preferred examples thereof include crosslinked vinyl alcohol polymers, modified vinyl alcohol polymers, polysaccharides, and combinations thereof. From the viewpoint of expanding the range of applications (e.g., use in applications requiring higher strength), the hydroxyl group-containing polymer is also preferably a hydroxyl group-containing polymer other than a hydrogel-forming hydroxyl group-containing polymer, and preferred examples thereof include ethylene-vinyl alcohol copolymers. From the viewpoint of being able to perform plasma treatment under reduced pressure conditions, plasma treatment is preferably performed on a hydroxyl group-containing polymer in a dry state. When the hydroxyl group-containing polymer has a shape such as particles (irregular particles or spherical particles), a fine molded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a cloth, a hollow fiber, a porous monolith, or a coated article, it is preferable to subject the hydroxyl group-containing polymer having such a shape to plasma treatment in the presence of a halogenated hydrocarbon gas.

[0053] <Method for preparing hydroxyl group-containing polymer> First, a method for preparing a hydroxyl group-containing polymer when the hydroxyl group-containing polymer is a crosslinked vinyl alcohol polymer will be described. The crosslinked vinyl alcohol polymer can be prepared, for example, by a method including a step of preparing an uncrosslinked polymer solution containing a vinyl alcohol polymer (uncrosslinked polymer solution preparation step), then optionally a step of molding the uncrosslinked polymer solution (molding step), and then a step of crosslinking the molded or unmolded vinyl alcohol polymer (crosslinking step).

[0054] (Uncrosslinked polymer solution preparation step) An example of a method for preparing a vinyl alcohol-based polymer is a method in which a polyvinyl ester obtained by polymerizing a vinyl ester-based monomer is saponified, and the ester groups in the polyvinyl ester are converted to hydroxyl groups.

[0055] Examples of the vinyl ester monomer include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These may be used alone or in combination of two or more. Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of preparation costs. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0056] Furthermore, the polyvinyl ester may contain structural units derived from monomers other than vinyl ester-based monomers, if necessary, within the range that does not impair the effects of the present invention.Examples of such monomers include α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or its salts; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or its salts; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, methyl ... Methacrylic acid alkyl esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidopropanesulfonic acid or a salt thereof, acrylamidopropyldimethylamine or a salt or quaternary salt thereof, N-methylolacrylamide, or derivatives thereof; methacrylamide derivatives such as methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or a salt thereof, methacrylamidepropyldimethylamine or a salt or quaternary salt thereof, N-methylolmethacrylamide or a derivative thereof; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether, and stearyl vinyl ether; nitriles such as acrylonitrile and methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylidene halides such as vinylidene chloride and vinylidene fluoride; allyl compounds such as allyl acetate and allyl chloride; maleic acid or a salt, ester, or acid anhydride thereof; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0057] When the polyvinyl ester contains structural units derived from monomers other than vinyl ester-based monomers, the content of the structural units derived from monomers other than vinyl ester-based monomers is preferably 20 mol % or less, more preferably 10 mol % or less, and even more preferably 5 mol % or less, based on the total structural units constituting the polyvinyl ester.

[0058] The method for saponifying the polyvinyl ester is not particularly limited, and can be the same as conventional methods. For example, alcoholysis or hydrolysis using an alkali or acid catalyst can be applied. Among them, saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferable.

[0059] An uncrosslinked polymer solution containing the vinyl alcohol polymer can be prepared by mixing the vinyl alcohol polymer described above with a solvent. The solvent is preferably one or more solvents selected from the group consisting of water and water-soluble organic solvents. It is more preferable that the solvent contains water, and even more preferable that the solvent is water. Examples of water-soluble organic solvents include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin.

[0060] The content of the solvent in the uncrosslinked polymer solution is preferably 50% by mass or more, more preferably 55% by mass or more, even more preferably 60% by mass or more, and is preferably 99.999% by mass or less, more preferably 99.99% by mass or less, even more preferably 99.9% by mass or less, based on the total mass of the uncrosslinked polymer solution. When the uncrosslinked polymer solution contains a water-soluble organic solvent, the content thereof is preferably 30% by mass or less, more preferably 20% by mass or less, even more preferably 10% by mass or less, based on the total mass of the uncrosslinked polymer solution.

[0061] From the viewpoint of sufficient mechanical strength or dimensions of the crosslinked body of the vinyl alcohol polymer, the content of the vinyl alcohol polymer in the uncrosslinked polymer solution is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, relative to the total mass of the uncrosslinked polymer solution. Furthermore, from the viewpoint of suppressing the increase in viscosity of the uncrosslinked polymer solution and obtaining good moldability, the content of the vinyl alcohol polymer in the uncrosslinked polymer solution is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less, relative to the total mass of the uncrosslinked polymer solution.

[0062] (Molding step) The prepared uncrosslinked polymer solution may be molded, and preferably molded. When molding the uncrosslinked polymer solution, the method is not particularly limited, and it can be molded into the shape of particles (irregular particles or spherical particles), fine molded bodies, arbitrary shaped articles molded by 3D printers, films, threads, cloths, hollow fibers, porous monoliths, coated articles, etc. by known methods.

[0063] The method for forming spherical particles is not particularly limited, and known techniques can be used. Examples include suspension polymerization, membrane emulsification, microfluidics, nozzle extrusion, and spray drying. In addition to these, submerged electrospray, as described in Biotechnology and Bioengineering, 2012, Vol. 109, pp. 1561-1570, can also be used.

[0064] The suspension polymerization method is a method in which an uncrosslinked polymer solution containing a radical polymerization initiator is used as the dispersed phase, and an incompatible liquid is used as the continuous phase in a water-in-oil liquid-liquid dispersion system to crosslink the uncrosslinked polymer and obtain spherical particles of a crosslinked vinyl alcohol polymer. An emulsion stabilizer may be added to the continuous phase as needed. The dispersed phase is broken down into minute droplets by mechanical energy (generally using a stirring blade), and crosslinking of the uncrosslinked polymer proceeds within the droplets.

[0065] The membrane emulsification method involves extruding an uncrosslinked polymer solution containing a radical polymerization initiator through a membrane with uniform pore size into the continuous phase to form uniform water-in-oil microdroplets, which are then crosslinked to obtain spherical particles of a crosslinked vinyl alcohol polymer. As with the suspension polymerization method, an emulsion stabilizer may be added to the continuous phase as needed. Examples of membranes with uniform pore size include SPG (Shirasu Porous Glass) membranes and hydrophilic or hydrophobic membranes with uniform pores manufactured by Micropore Technologies. For example, the basic method described in Journal of Membrane Science, 2017, Vol. 524, pp. 79-86, can be used.

[0066] The microfluidic method involves flowing the continuous phase through a microchannel on the order of micrometers, injecting a dispersed phase, an uncrosslinked polymer solution containing a radical polymerization initiator, into the continuous phase through a microchannel on the order of micrometers to form microdroplets, and crosslinking the uncrosslinked polymer to obtain spherical particles of a crosslinked vinyl alcohol polymer. Depending on the method of injecting the dispersed phase into the continuous phase, devices are classified into terrace-like devices, T-junctions, flow-focusing microchannel devices (FFDs), capillary devices (parallel flow, cross-flow, FFD), and the like. For example, methods such as those described in Chemical Engineering and Technology, 2008, Vol. 31, pp. 1099-1115, can be applied. Even in the microfluidic method, an emulsion stabilizer may be added to the continuous phase as needed.

[0067] Examples of the emulsion stabilizer that can be used include water-soluble polymers such as partially saponified polyvinyl alcohol, gelatin, hydroxymethyl cellulose, methyl cellulose, and carboxymethyl cellulose, and surfactants such as sodium dodecylbenzenesulfonate, sorbitan monooleate, dioctyl sulfosuccinate, polyoxyethylene sorbitan monooleate (Tween-80), and polyoxyethylene sorbitan monostearate (Tween-60).

[0068] Commonly used liquids that are incompatible with the dispersed phase include, for example, water-insoluble oils, toluene, hexane, octane (including isooctane), liquid paraffin, edible oils, and dichloroethane.

[0069] The nozzle extrusion method is a method in which an uncrosslinked polymer solution containing a radical polymerization initiator is extruded through a nozzle into air and dropped onto the continuous phase to form water-in-oil microdroplets, which crosslink the uncrosslinked polymer and produce spherical particles of crosslinked vinyl alcohol-based polymer. To efficiently generate droplets, the uncrosslinked polymer solution can be mechanically cut, for example, by vibrating the nozzle or using a rotating disk or rotating nozzle. More specific examples of the nozzle extrusion method include those described in Chemical Papers, 2008, Vol. 62, pp. 364-374. In the nozzle extrusion method, the uncrosslinked polymer is crosslinked while the droplets are falling in the air, thereby producing spherical particles of crosslinked vinyl alcohol-based polymer without using the continuous phase.

[0070] In the nozzle extrusion method, for example, alginic acid or the like can be dissolved in an uncrosslinked polymer solution containing a radical polymerization initiator, and then the solution can be dropped into a polyvalent metal ion solution by the nozzle extrusion method to form spherical particles. This method utilizes the gelling property of alginic acid due to polyvalent metal ions, and by crosslinking the uncrosslinked polymer after forming the spherical particles using the method described below, the crosslinked vinyl alcohol polymer of the present invention can be obtained.

[0071] In the spray drying method, an uncrosslinked polymer solution is atomized through a nozzle, a rotating disk, or the like to form fine droplets, which are then dried by heat to obtain spherical particles. When a photoradical polymerization initiator is used, crosslinking of the uncrosslinked polymer can be carried out by irradiating the fine droplets with light at the stage when they are formed, or when a thermal radical polymerization initiator is used, by drying the fine droplets with heat. Of course, after obtaining the dried spherical particles, they may be crosslinked by applying light or heat depending on the type of radical polymerization initiator to obtain a crosslinked vinyl alcohol polymer of the present invention.

[0072] Any of these particle production methods can be suitably used as a method for forming spherical particles of a crosslinked vinyl alcohol polymer. From the viewpoint of controlling the average particle size of the crosslinked vinyl alcohol polymer, suspension polymerization using a static mixer, membrane emulsification, microfluidization, nozzle extrusion, and spray drying are preferred, with membrane emulsification, microfluidization, and nozzle extrusion being more preferred. These methods are basically characterized by the ability to strictly define the volume of microdroplets using a fine hole or nozzle, which contributes to controlling the average particle size. Of course, it is also a preferred method to control the average particle size of the obtained spherical particles by classifying spherical particles having a particle size distribution produced by suspension polymerization, which converts the particles into microdroplets using a stirring blade. Note that suspension polymerization is preferred from the viewpoint of productivity of the crosslinked vinyl alcohol polymer.

[0073] A film-like crosslinked vinyl alcohol polymer can be formed by pouring an uncrosslinked polymer solution onto a glass plate or the like sandwiched between spacers. As a more industrial method, a film-like crosslinked vinyl alcohol polymer can be formed by subjecting the uncrosslinked polymer solution to an extrusion molding method such as a T-die method, or a solution casting method. The solution casting method is a method in which an uncrosslinked polymer solution is poured onto a smooth casting drum or stainless steel belt and spread to form a film. A film-like crosslinked vinyl alcohol polymer can be obtained by crosslinking the film-like uncrosslinked polymer solution, or by evaporating the solvent from the film-like uncrosslinked polymer solution to obtain a dried film and then crosslinking the uncrosslinked polymer.

[0074] The crosslinked vinyl alcohol polymer filaments can be formed by a so-called wet spinning method. That is, a solution of an uncrosslinked polymer is extruded through a nozzle into a coagulation bath of dimethyl sulfoxide, a saturated aqueous sodium sulfate solution, a saturated aqueous ammonium sulfate solution, or the like to form filaments, which are then crosslinked as they are or after drying, thereby obtaining the crosslinked vinyl alcohol polymer of the present invention.

[0075] Examples of methods for coating a substrate with a crosslinked vinyl alcohol polymer to form a coated article include methods of coating a substrate with an uncrosslinked polymer solution using a roll coater such as a gravure coater, reverse coater, slot die coater, lip coater, knife coater, or comma coater; a spin coater, dip coater, spray coater, or inkjet coater. Furthermore, coating a spherical particle substrate with a crosslinked vinyl alcohol polymer can be carried out by coating the spherical particle substrate with an uncrosslinked polymer solution using, for example, a fluidized bed granulation coater. The optimal method from the above molding methods can be freely selected depending on the shape of the desired crosslinked vinyl alcohol polymer.

[0076] (Crosslinking Step) The crosslinking step may be performed while the molded or unmolded vinyl alcohol polymer (hereinafter also referred to as molded / unmolded vinyl alcohol polymer) still contains the solvent, or may be performed after removing the solvent from the molded / unmolded vinyl alcohol polymer. If the molded / unmolded vinyl alcohol polymer is crosslinked while still containing the solvent, shrinkage may be severe in the optional drying step described below, making it difficult to form the polymer into shapes such as films, threads, cloths, hollow fibers, and coatings. In this case, it is preferable to perform crosslinking after removing the solvent from the molded / unmolded vinyl alcohol polymer by drying. The drying method may be the same as the method described in the optional drying step described below. The degree of drying may be appropriately selected depending on the composition or shape of the molded / unmolded vinyl alcohol polymer or the crosslinking method. From the viewpoint of shrinkage in the optional drying step described below, the solvent content, as represented by the following formula (1), is preferably 50% by mass or less, more preferably 25% by mass or less, and even more preferably 10% by mass or less. Solvent content [%] = [(mass of solvent contained in molded / unmolded vinyl alcohol-based polymer) / (mass of molded / unmolded vinyl alcohol-based polymer in dry state + mass of solvent contained in molded / unmolded vinyl alcohol-based polymer)] × 100 Formula (1) The solvent content can be measured by a method similar to that described in the drying step described below.

[0077] The vinyl alcohol polymer may be physically crosslinked or may be crosslinked by a covalent bond. From the viewpoint of controlling the physical properties of the resulting crosslinked vinyl alcohol polymer and / or from the viewpoint of increasing the stability of the crosslinked vinyl alcohol polymer during swelling, crosslinking by a covalent bond is preferred.

[0078] Examples of methods for physically crosslinking a vinyl alcohol polymer include freezing and thawing an aqueous solution of the vinyl alcohol polymer, or dissolving the vinyl alcohol polymer in a mixed solvent of dimethyl sulfoxide and water by heating and cooling the solution to room temperature. A specific example of a method for freezing and thawing an aqueous solution of the vinyl alcohol polymer includes freezing the aqueous solution of the vinyl alcohol polymer at -20°C for 15 hours or more and then thawing it at 4°C for 8 hours or more, repeating this freeze-thaw process three or more times.

[0079] Examples of a method for crosslinking a vinyl alcohol polymer by a covalent bond include a method using a polyfunctional crosslinking agent that reacts with a side chain hydroxyl group of the vinyl alcohol polymer, and a method in which a functional group is introduced into the vinyl alcohol polymer by copolymerization or post-modification, and then the functional group is reacted.

[0080] Examples of methods using a polyfunctional crosslinking agent that reacts with the side chain hydroxyl groups of a vinyl alcohol polymer include a method of reacting a polyfunctional aldehyde compound such as glyoxal, malondialdehyde, or glutaraldehyde with a vinyl alcohol polymer under acidic conditions (polyacetal crosslinking), a method of reacting a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether with a vinyl alcohol polymer under alkaline conditions (polyether crosslinking), or a method of reacting a polyfunctional carboxylic acid compound such as maleic acid or succinic acid with a vinyl alcohol polymer (polyester crosslinking). From the viewpoint of obtaining a desirable degree of swelling and / or mechanical strength of the crosslinked vinyl alcohol polymer, the binding amount of the polyfunctional crosslinking agent that has undergone the crosslinking reaction with the vinyl alcohol polymer is preferably 0.01 to 20 mol%, more preferably 0.1 to 10 mol%, and even more preferably 0.5 to 5 mol%, based on the structural units derived from vinyl alcohol.

[0081] A method for introducing functional groups into a vinyl alcohol polymer by copolymerization and reacting the functional groups includes copolymerizing a vinyl ester monomer with a monomer other than the vinyl ester monomer that has a reactive substituent other than a hydroxyl group during the preparation of the vinyl alcohol polymer, followed by saponification to obtain a copolymerized modified polyvinyl alcohol (hereinafter sometimes abbreviated as "copolymerized modified PVA"), followed by crosslinking with a multifunctional crosslinking agent that reacts with functional groups such as carboxyl groups or amino groups present in the copolymerized modified PVA. From the viewpoint of obtaining a desirable degree of swelling and / or mechanical strength of the crosslinked vinyl alcohol polymer, the amount of the monomer having a reactive substituent other than a hydroxyl group relative to the total structural units of the vinyl alcohol polymer is preferably 0.01 to 20 mol%, more preferably 0.1 to 10 mol%, and even more preferably 0.5 to 5 mol%. In the following description, copolymer-modified PVA having a carboxy group may be referred to as "carboxylic acid-modified PVA," and copolymer-modified PVA having an amino group may be referred to as "amino-modified PVA."

[0082] Examples of monomers other than vinyl ester monomers that constitute carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, and itaconic acid; (meth)acrylic acid alkyl esters such as methyl (meth)acrylate and ethyl (meth)acrylate; α,β-unsaturated carboxylic acid anhydrides such as maleic anhydride and itaconic anhydride, and derivatives thereof. In this specification, (meth)acrylic acid means acrylic acid or methacrylic acid. A crosslinked carboxylic acid-modified PVA can be prepared by copolymerizing a vinyl ester monomer with a monomer other than the vinyl ester monomers described above, followed by saponification to prepare a carboxylic acid-modified PVA, and then crosslinking the carboxylic acid-modified PVA with a polyfunctional crosslinking agent that reacts with the introduced carboxyl groups, such as a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether, or by crosslinking the carboxylic acid-modified PVA with a polyfunctional amino compound such as ethylenediamine, polyethyleneimine, or polyallylamine in combination with a carbodiimide condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. From the viewpoint of obtaining a desirable degree of swelling and / or mechanical strength of the crosslinked vinyl alcohol polymer, the amount of the polyfunctional crosslinking agent relative to the carboxylic acid-modified PVA is preferably 0.01 to 20 mol %, more preferably 0.1 to 10 mol %, and even more preferably 0.5 to 5 mol %, based on the total structural units of the vinyl alcohol polymer. The amount of the carbodiimide condensing agent relative to the carboxy groups of the carboxylic acid-modified PVA is preferably 0.1 to 300 mol %, more preferably 1 to 200 mol %, and even more preferably 10 to 100 mol %. As will be described later, when preparing a crosslinked vinyl alcohol polymer in which a cell adhesion factor, a cell-interacting protein, or the like is conjugated to the crosslinked vinyl alcohol polymer, the carboxy group introduced into the carboxylic acid-modified PVA can be covalently bonded [amide bond (—CONH—)] to an amino group of the cell adhesion factor, the cell-interacting protein, or the like to prepare the crosslinked vinyl alcohol polymer.

[0083] A crosslinked amino-modified PVA can be prepared by copolymerizing a vinyl ester monomer with N-vinylformamide or the like, followed by saponification to prepare an amino-modified PVA, and then crosslinking the amino-modified PVA using a combination of a polyfunctional crosslinking agent (e.g., a polyfunctional epoxy compound, a polyfunctional carboxylic acid compound such as succinic acid or maleic acid) that reacts with the introduced amino groups and the carbodiimide condensing agent. From the viewpoint of obtaining a desirable degree of swelling and / or mechanical strength of the crosslinked vinyl alcohol polymer, the amount of the polyfunctional crosslinking agent relative to the amino-modified PVA is preferably 0.01 to 20 mol %, more preferably 0.1 to 10 mol %, and even more preferably 0.2 to 5 mol %, based on the total structural units of the vinyl alcohol polymer. The amount of the carbodiimide condensing agent relative to the amino groups of the amino-modified PVA is preferably 0.1 to 300 mol %, more preferably 1 to 200 mol %, and even more preferably 10 to 100 mol %.

[0084] An example of a method for introducing functional groups into a vinyl alcohol polymer by post-modification and then reacting the functional groups is a method for introducing ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer. The introduced ethylenically unsaturated groups easily react by adding an additive such as a radical initiator to induce a polymerization reaction, thereby preparing a crosslinked vinyl alcohol polymer. Furthermore, when a vinyl alcohol polymer contains ethylenically unsaturated groups, various molding methods, including 3D printing, become possible. Therefore, in a preferred embodiment, the vinyl alcohol polymer contains ethylenically unsaturated groups. The ethylenically unsaturated groups are preferably radically polymerizable groups, and specific examples include vinyl groups, (meth)acryloyl groups, (meth)acryloylamino groups; cyclic unsaturated hydrocarbon groups such as vinylphenyl groups, cyclohexenyl groups, cyclopentenyl groups, norbornenyl groups, and dicyclopentenyl groups; and derivatives thereof. The presence of ethylenically unsaturated groups in a vinyl alcohol polymer can be confirmed, for example, by nuclear magnetic resonance analysis (NMR).

[0085] The ethylenically unsaturated group is preferably introduced via a side chain or a terminal functional group of the vinyl alcohol polymer, and more preferably by reacting a hydroxyl group on the side chain of the vinyl alcohol polymer with a compound containing an ethylenically unsaturated group (hereinafter, sometimes abbreviated as "ethylenically unsaturated group-containing compound").

[0086] Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include (meth)acrylic acid or its derivatives such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, (meth)acrylic acid ester, etc. These compounds are subjected to an esterification reaction or an ester exchange reaction, preferably in the presence of a base, to introduce a (meth)acryloyl group into the vinyl alcohol polymer.

[0087] Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer also include compounds containing an ethylenically unsaturated group and a glycidyl group in the molecule, specifically glycidyl (meth)acrylate, allyl glycidyl ether, etc. By subjecting these compounds to an etherification reaction in the presence of a base, it is possible to introduce a (meth)acryloyl group or an allyl group into the vinyl alcohol polymer.

[0088] Examples of ethylenically unsaturated group-containing compounds to be reacted with the 1,3-diol group of a vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule, such as acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2-carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde. By subjecting these compounds to an acetalization reaction in the presence of an acid catalyst, an ethylenically unsaturated group can be introduced into the vinyl alcohol polymer. More specifically, by subjecting, for example, 5-norbornene-2-carboxaldehyde, 3-vinylbenzaldehyde, or 4-vinylbenzaldehyde to an acetalization reaction, a norbornenyl group or a vinylphenyl group can be introduced into the vinyl alcohol polymer. Furthermore, a (meth)acryloylamino group can be introduced into a vinyl alcohol polymer by reacting with N-(2,2-dimethoxyethyl)(meth)acrylamide, etc. Methods for introducing an ethylenically unsaturated group into a vinyl alcohol polymer include reactions other than those exemplified above, and a combination of two or more reactions may be used.

[0089] Other methods for introducing the ethylenically unsaturated group include reacting a reactive substituent, such as a carboxy group present in a carboxylic acid-modified PVA or an amino group present in an amino-modified PVA, with an ethylenically unsaturated group-containing compound. The carboxy group of a carboxylic acid-modified PVA can be reacted with, for example, glycidyl methacrylate under acidic conditions to form an ester bond, thereby introducing a methacryloyl group into the carboxylic acid-modified PVA. The amino group of an amino-modified PVA can be introduced with an acryloylamino group by, for example, amidation reaction with acrylic anhydride in the presence of a base, or with a vinyloxycarbonyl group by amidation reaction with divinyl adipate. Methods for introducing an ethylenically unsaturated group via a copolymer-modified PVA include reactions other than those exemplified above, and a combination of two or more reactions may be used.

[0090] From the viewpoint of ease of preparation, the vinyl alcohol polymer having an ethylenically unsaturated group is preferably a vinyl alcohol polymer in which an ethylenically unsaturated group has been introduced via a side chain hydroxyl group or a side chain hydroxyl group such as a 1,3-diol group, and more preferably a vinyl alcohol polymer in which a side chain hydroxyl group has been subjected to an esterification reaction or transesterification reaction with (meth)acrylic acid or a derivative thereof, or a vinyl alcohol polymer in which a 1,3-diol group has been subjected to an acetalization reaction with a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule.

[0091] The introduction rate of the ethylenically unsaturated group is preferably 10 mol% or less, more preferably 5 mol% or less, and even more preferably 3 mol% or less, based on all structural units constituting the vinyl alcohol polymer, from the viewpoint of suppressing embrittlement of the crosslinked vinyl alcohol polymer. Also, from the viewpoint of accelerating the crosslinking reaction, rapidly forming a crosslinked vinyl alcohol polymer, and improving the elastic modulus of the obtained crosslinked vinyl alcohol polymer, it is preferably 0.01 mol% or more, more preferably 0.1 mol% or more, and even more preferably 0.5 mol% or more.

[0092] From the viewpoint of improving the mechanical strength of the crosslinked vinyl alcohol polymer and introducing a functional group other than a hydroxyl group, the vinyl alcohol polymer having an ethylenically unsaturated group may further contain a structural unit derived from another monomer. Examples of the other monomer include acrylamides such as acrylamide, N-isopropylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and N,N-dimethylacrylamide; α,β-unsaturated carboxylic acids such as (meth)acrylic acid, crotonic acid, itaconic acid, maleic acid, and fumaric acid; water-soluble radically polymerizable monomers such as vinylpyridine, hydroxyethyl(meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; and compounds having two or more ethylenically unsaturated groups in the molecule such as N,N'-methylenebisacrylamide, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate. From the viewpoint of improving the mechanical strength of the crosslinked vinyl alcohol polymer, the content of the other monomer is preferably 50% by mass or less, more preferably 30% by mass or less, and even more preferably 10% by mass or less, based on the total mass of the vinyl alcohol polymer having an ethylenically unsaturated group.

[0093] When active energy rays or heat are applied to a vinyl alcohol polymer having an ethylenically unsaturated group, the ethylenically unsaturated group in the vinyl alcohol polymer crosslinks, thereby obtaining a crosslinked vinyl alcohol polymer. Examples of active energy rays include gamma rays, ultraviolet rays, visible light, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron beams, plasma flow, ionizing rays, and particle beams. When the vinyl alcohol polymer is crosslinked by ultraviolet rays, visible light, infrared rays (heat rays), or heat among the active energy rays, it is preferable that the uncrosslinked polymer solution contains a radical polymerization initiator. Examples of radical polymerization initiators include photoradical polymerization initiators and thermal radical polymerization initiators.

[0094] The photoradical polymerization initiator is not particularly limited as long as it initiates radical polymerization by irradiation with active energy rays such as ultraviolet light or visible light. From the viewpoint of being able to wash away the photoradical polymerization initiator after crosslinking, it is preferable that the photoradical polymerization initiator be water-soluble. Specific examples of the photoradical polymerization initiator include 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (trade name "Omnirad 2959", manufactured by IGM RESINS B.V.), α-ketoglutaric acid, phenyl(2,4,6-trimethylbenzoyl)phosphinic acid lithium salt (trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.), and Eosin Y. When a photoradical polymerization initiator is used, the amount thereof is typically 0.0001 to 10 parts by mass, preferably 0.001 to 5 parts by mass, and more preferably 0.005 to 3 parts by mass, per 100 parts by mass of the vinyl alcohol-based polymer.

[0095] The thermal radical polymerization initiator is not particularly limited as long as it initiates radical polymerization by heat. Examples include azo initiators and peroxide initiators that are commonly used in radical polymerization. When a thermal radical polymerization initiator is used, the amount thereof is usually 0.001 to 30 parts by mass, preferably 0.01 to 10 parts by mass, and more preferably 0.1 to 5 parts by mass, per 100 parts by mass of the vinyl alcohol polymer.

[0096] The azo initiator is preferably water-soluble from the viewpoint of being able to be washed away after crosslinking. Specific examples thereof include 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (trade name "VA-044"), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate (trade name "VA-044B"), 2,2'-azobis[2-methylpropionamidine]dihydrochloride (trade name "V-50"), 2,2'-azobis[N-(2-carboxyethyl)- ... Examples of suitable azobis[2-(2-methylpropionamidine) tetrahydrate (trade name "VA-057"), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (trade name "VA-061"), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (trade name "VA-086"), and 4,4'-azobis(4-cyanopentanoic acid) (trade name "V-501") (all manufactured by Wako Pure Chemical Industries, Ltd.) are listed.

[0097] As the peroxide initiator, a peroxide initiator that does not generate gas is preferred from the viewpoint of improving the physical properties of the vinyl alcohol polymer, and a water-soluble peroxide initiator is preferred from the viewpoint of being able to wash away the thermal radical polymerization initiator after crosslinking. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate, and sodium persulfate.

[0098] Alternatively, a redox polymerization initiator, which is a combination of a peroxide initiator and a reducing agent, may be used. Known reducing agents can be used as the reducing agent. Examples of preferred reducing agents include highly water-soluble N,N,N',N'-tetramethylethylenediamine, sodium sulfite, sodium hydrogensulfite, and sodium hydrosulfite. When a redox polymerization initiator is used, the amount thereof is preferably 0.001 to 30 parts by mass, more preferably 0.01 to 10 parts by mass, and even more preferably 0.1 to 5 parts by mass, per 100 parts by mass of the vinyl alcohol polymer.

[0099] When crosslinking a vinyl alcohol polymer having a vinyl group as the ethylenically unsaturated group, for example, a polythiol having two or more thiol groups in the molecule may be added to promote crosslinking, and crosslinking may be carried out using a thiol-ene reaction. The polythiol is preferably water-soluble, and examples thereof include polythiols having a hydroxyl group such as dithiothreitol; and polythiols containing an ether bond, such as terminal thiolated products such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol. When a polythiol is used, the amount thereof is preferably 0.01 to 20 mol %, more preferably 0.1 to 10 mol %, and even more preferably 0.5 to 5 mol %, based on the total structural units of the vinyl alcohol polymer.

[0100] The method for crosslinking a vinyl alcohol polymer by a covalent bond can be appropriately selected from the methods exemplified above. From the viewpoints of the toxicity of the unreacted crosslinking agent or condensing agent contained in the crosslinked vinyl alcohol polymer and ease of molding, a method in which a functional group is introduced into the vinyl alcohol polymer by post-modification and the functional group is reacted, in particular a method in which an ethylenically unsaturated group is introduced into the side chain of the vinyl alcohol polymer by post-modification, is preferred.

[0101] When a molding step is performed, the uncrosslinked polymer solution may contain, in addition to the vinyl alcohol polymer, components necessary for crosslinking, such as the crosslinking agent or initiator, before the molding step. When the molded / unmolded vinyl alcohol polymer is dried as described above, the components necessary for crosslinking may be added before drying. As described above, optimal crosslinking conditions can be adopted depending on the vinyl alcohol polymer or crosslinking method. However, since molding after crosslinking is difficult, molding is preferably performed before crosslinking is completed. When the components necessary for crosslinking are mixed with the uncrosslinked polymer solution before the molding step, it is preferable to mix them immediately before the molding step. Furthermore, when a cell adhesion factor or cell-interacting protein, etc. is contained before crosslinking of the vinyl alcohol polymer and crosslinking requires heat, the temperature of the crosslinking reaction is preferably 100°C or less, more preferably 60°C or less, and even more preferably 37°C or less, from the viewpoint of maintaining the activity of the cell adhesion factor or cell-interacting protein, etc.

[0102] After the crosslinking step, the crosslinked vinyl alcohol polymer may be washed with a washing solution, if necessary. When crosslinking is performed using a water-soluble photoradical polymerization initiator or a thermal radical polymerization initiator, the photoradical polymerization initiator or the thermal radical polymerization initiator can be removed by washing. Washing can be performed by a conventional method, and may be performed once or multiple times. Examples of the washing solution include one or more selected from the group consisting of water and the water-soluble organic solvents, with water being preferred. When the washing solution is water, a buffer solution, such as a phosphate buffer, a Tris buffer, a HEPES buffer, an acetate buffer, a citrate buffer, a bicarbonate buffer, or an MES buffer, may be used in combination.

[0103] <Ethylene-vinyl alcohol copolymer> Next, a method for preparing a hydroxyl group-containing polymer when the hydroxyl group-containing polymer is an ethylene-vinyl alcohol copolymer will be described. The ethylene-vinyl alcohol copolymer can be prepared, for example, by a method including a step of preparing an ethylene-vinyl alcohol copolymer by a method including saponifying a copolymer of ethylene and a vinyl ester (ethylene-vinyl alcohol copolymer preparation step), and then, optionally, a step of molding the ethylene-vinyl alcohol copolymer (molding step).

[0104] (Ethylene-Vinyl Alcohol Copolymer Preparation Process) The preparation and saponification of a copolymer of ethylene and a vinyl ester can be carried out by known methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization, and may be carried out either continuously or batchwise. Examples of vinyl esters include vinyl acetate, vinyl formate, vinyl propionate, vinyl valerate, vinyl caprate, vinyl laurate, vinyl stearate, vinyl pivalate, vinyl versatate, and other aliphatic carboxylic acid vinyl esters, with vinyl acetate being preferred. As described above, when the ethylene-vinyl alcohol copolymer has structural units other than ethylene units, vinyl alcohol units, and vinyl ester units, an ethylene-vinyl alcohol copolymer having such structural units can be prepared by using a monomer corresponding to the structural unit. As an example, the polymerization conditions for batchwise solution polymerization will be described below, but the method for preparing the ethylene-vinyl ester copolymer of the present invention is not limited thereto.

[0105] As the solvent, alcohols are preferred, but other organic solvents capable of dissolving ethylene, vinyl esters, and ethylene-vinyl ester copolymers (e.g., dimethyl sulfoxide) can also be used. Examples of preferred alcohols include methyl alcohol, ethyl alcohol, propyl alcohol, n-butyl alcohol, and t-butyl alcohol, with methyl alcohol being particularly preferred. It is preferable to use the solvent in an amount such that the proportion of the ethylene-vinyl alcohol copolymer in the solution after polymerization is preferably 5 to 85% by mass, more preferably 20 to 70% by mass. The ratio of ethylene to the other monomers may be appropriately adjusted so that the ethylene content and vinyl ester content, etc., in the resulting copolymer are the desired values.

[0106] Known catalysts may also be used. Examples include azonitrile initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis-(2,4-dimethylvaleronitrile), 2,2'-azobis-(4-methyl-2,4-dimethylvaleronitrile), and 2,2'-azobis-(2-cyclopropylpropionitrile), and organic peroxide initiators such as isobutyryl peroxide, cumyl peroxyneodecanoate, diisopropyl peroxycarbonate, di-n-propyl peroxydicarbonate, t-butyl peroxyneodecanoate, lauroyl peroxide, benzoyl peroxide, and t-butyl hydroperoxide. The polymerization temperature is preferably 20 to 90°C, more preferably 40 to 70°C. The polymerization rate is usually 10 to 90% by mass, preferably 30 to 80% by mass, based on the vinyl ester charged. The polymerization time is usually 2 to 15 hours, preferably 3 to 11 hours.

[0107] After a predetermined period of polymerization or after a predetermined conversion has been reached, it is preferable to add a polymerization inhibitor as needed, remove unreacted ethylene gas by evaporation, and then purge the unreacted vinyl ester from the reaction system. Examples of a method for purging the unreacted vinyl ester from the ethylene-vinyl ester copolymer from which ethylene has been removed by evaporation include a method in which an ethylene-vinyl ester copolymer solution is continuously fed at a constant rate from the top of a column packed with Raschig rings, organic solvent vapor such as methanol is blown into the bottom of the column, a mixed vapor of the organic solvent such as methanol and the unreacted vinyl ester is discharged from the top of the column, and the ethylene-vinyl ester copolymer solution from which the unreacted vinyl ester has been removed is taken out from the bottom of the column.

[0108] The vinyl ester component in the ethylene-vinyl ester copolymer can be saponified by adding an alkali catalyst to the ethylene-vinyl ester copolymer solution from which unreacted vinyl ester has been removed. The saponification method may be either continuous or batchwise. Examples of alkali catalysts that can be used include sodium hydroxide, potassium hydroxide, and alkali metal alcoholates. An example of saponification conditions for the batchwise method is shown below, but the saponification conditions can be adjusted as desired depending on the type of ethylene-vinyl ester copolymer, the degree of saponification, and other factors. Concentration of the ethylene-vinyl ester copolymer solution: preferably 10 to 50% by mass, more preferably 20 to 40% by mass. Amount of alkali catalyst: preferably 0.02 to 0.6 equivalents, more preferably 0.03 to 0.6 equivalents per vinyl ester component. Reaction temperature: preferably 30 to 60°C, more preferably 35 to 55°C. Reaction time: typically 1 to 6 hours, preferably 1.5 to 5 hours.

[0109] After the saponification reaction, it is preferable to neutralize the reaction mixture as necessary and then wash it to remove the alkali catalyst, by-product salts, and other impurities. For washing, acids such as water, phosphoric acid, pyrophosphoric acid, phosphorous acid, oxalic acid, succinic acid, adipic acid, tartaric acid, citric acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and acetic acid, or polybasic acids and their salts, as well as mixtures of two or more thereof, can be used. Among these, a method in which the ethylene-vinyl ester copolymer is immersed in an aqueous solution of the compound and then drained is preferred. This washing method may be performed by either a batch method or a continuous method, and may be performed once or multiple times. Furthermore, prior to this washing step, a molding step (described below) may be performed, and the resulting molded body may be washed.

[0110] (Molding step) The prepared ethylene-vinyl alcohol copolymer may be molded, and is preferably molded. When the ethylene-vinyl alcohol copolymer is molded, the method is not particularly limited, and it can be molded by a known method into shapes such as particles (irregular particles or spherical particles), fine molded bodies, arbitrarily shaped articles molded with a 3D printer, films, threads, fabrics (e.g., woven fabrics, knitted fabrics, and nonwoven fabrics), hollow fibers, porous monoliths, coated articles, and the like.

[0111] For example, when a nonwoven fabric is obtained using an ethylene-vinyl alcohol copolymer, steam jet technology is used to blow heated steam at high speed onto resin fibers to bond the fibers together, thereby obtaining a nonwoven fabric.

[0112] <Plasma Treatment> Halogen can be introduced into a hydroxyl-containing polymer by plasma treatment. The plasma treatment can be any treatment capable of irradiating the surface of a hydroxyl-containing polymer with plasma in the presence of a halogenated hydrocarbon gas. Examples of plasma treatment include atmospheric pressure plasma treatment, in which gas flowing between opposing electrodes is sequentially converted to a plasma state by discharge between the electrodes at atmospheric pressure or near atmospheric pressure, and the resulting plasma is then irradiated onto the hydroxyl-containing polymer. Also included is low-pressure plasma treatment, in which gas flowing between opposing electrodes is sequentially converted to a plasma state by discharge between the electrodes at low pressure (i.e., 500 Pa or less), preferably 30 to 60 Pa, more preferably 40 to 50 Pa, and the resulting plasma is then irradiated onto the hydroxyl-containing polymer. Plasma treatment can modify at least a portion of the surface of the hydroxyl-containing polymer, imparting cell adhesive properties, and can decompose the halogenated hydrocarbon gas, thereby introducing the resulting halogen into the hydroxyl-containing polymer.

[0113] In atmospheric pressure plasma treatment, the irradiation intensity of the discharge between electrodes when generating plasma is preferably 50 W or more, more preferably 75 W or more, and even more preferably 100 W or more, from the viewpoint of generating a sufficient amount of plasma. The upper limit of the irradiation intensity is not particularly limited, but is generally preferably 1000 W or less. The treatment temperature is preferably 0 to 40°C, more preferably 15 to 30°C, from the viewpoint of ensuring sufficient reactivity and suppressing denaturation due to overheating. The treatment time is preferably 5 minutes or more, more preferably 10 minutes or more, and even more preferably 20 minutes or more. The upper limit of the treatment time is not particularly limited, but is generally 1 hour or less.

[0114] In low-pressure plasma treatment, the power (irradiation intensity) discharged between electrodes when generating plasma is preferably 50 W or more, more preferably 75 W or more, and even more preferably 90 W or more, from the viewpoint of generating a sufficient amount of plasma. The upper limit of the irradiation intensity is not particularly limited, but is generally preferably 1000 W or less. The treatment temperature is preferably 0 to 40°C, more preferably 15 to 30°C, from the viewpoint of ensuring sufficient reactivity and suppressing denaturation due to overheating. The treatment time is preferably 5 minutes or more, more preferably 10 minutes or more, and even more preferably 20 minutes or more. The upper limit of the treatment time is not particularly limited, but is generally 1.5 hours or less.

[0115] Low-pressure plasma treatment is preferred from the viewpoint of being able to remove impurities generated during plasma treatment. For example, if impurities adhere to a hydroxyl group-containing polymer, the impurities are vaporized by plasma treatment. In low-pressure plasma treatment, the vaporized impurities are sucked into a vacuum pump (or trapped if a trap is provided) and removed. Therefore, the influence of impurities on a hydroxyl group-containing polymer treated by low-pressure plasma treatment is reduced, which is advantageous in terms of stabilizing physical properties.

[0116] Examples of halogenated hydrocarbon gases include fluorocarbon gases such as carbon tetrafluoride, carbon hexafluoride, and propylene hexafluoride; and halogenated hydrocarbon gases such as carbon monochloride trifluoride. Carbon tetrafluoride is preferred from the viewpoint of availability. The halogenated hydrocarbon gases can be used alone or in combination.

[0117] The flow rate of the halogenated hydrocarbon gas is preferably 1 to 10,000 cc / min, more preferably 5 to 5,000 cc / min, even more preferably 8 to 3,000 cc / min, and particularly preferably 10 to 2,000 cc / min, from the viewpoint of being able to introduce the desired amount of halogen into the hydroxyl group-containing polymer. A gas other than a halogenated hydrocarbon gas may also be passed through. Examples of such a gas include argon gas, nitrogen gas, helium gas, oxygen gas, hydrogen gas, and mixtures of two or more thereof. When a gas other than a halogenated hydrocarbon gas is used, the proportion of the gas other than a halogenated hydrocarbon gas is preferably 5 to 95%, more preferably 7 to 90%, and particularly preferably 10 to 85%, based on the total volume of the gas passed through.

[0118] It is preferable to irradiate the hydroxyl group-containing polymer with plasma while moving it so that the entire hydroxyl group-containing polymer is irradiated with plasma. For example, when irradiating a particulate hydroxyl group-containing polymer with plasma, the entire hydroxyl group-containing polymer can be irradiated with plasma by irradiating the hydroxyl group-containing polymer with plasma while rotating the container containing the hydroxyl group-containing polymer.

[0119] <Drying Step> Plasma treatment is preferably performed on a dried hydroxyl-containing polymer, as it can be performed under reduced pressure. Therefore, if necessary, it is preferable to dry the hydroxyl-containing polymer before plasma treatment. The drying method for the hydroxyl-containing polymer is not particularly limited, and common methods such as hot air drying, ventilation drying, vacuum drying, reduced pressure drying, and freeze drying can be used. In addition, the hydroxyl-containing polymer can be immersed in a volatile water-soluble organic solvent to remove components such as water contained in the hydroxyl-containing polymer, followed by drying. Examples of volatile water-soluble organic solvents include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and acetone. Drying conditions can be appropriately selected. The water content of the hydroxyl-containing polymer after drying, as represented by the following formula (2), is preferably 40% by mass or less, more preferably 30% by mass or less, and even more preferably 25% by mass or less. Water content [%] = [(mass of water contained in the hydroxyl group-containing polymer) / (mass of the hydroxyl group-containing polymer in a dry state + mass of water contained in the hydroxyl group-containing polymer)] × 100 Formula (2)

[0120] The appropriate method for measuring the water content of a hydroxyl-containing polymer varies depending on the type of solvent contained in the hydroxyl-containing polymer. When the hydroxyl-containing polymer contains only water as a solvent, the water content of the hydroxyl-containing polymer can be calculated by directly evaporating the water contained in the hydroxyl-containing polymer using a heat-drying moisture meter and measuring the mass of the remaining dry hydroxyl-containing polymer. When the hydroxyl-containing polymer contains water and the water-soluble organic solvent, the hydroxyl-containing polymer can be calculated by directly evaporating the water contained in the hydroxyl-containing polymer using a heat-drying moisture meter and measuring the mass of the remaining dry hydroxyl-containing polymer. １ The water content can be measured by H-NMR measurement. Specifically, the hydroxyl group-containing polymer is swollen with deuterated DMSO, １ The content of water and the water-soluble organic solvent can be calculated by determining the H-NMR spectrum and the integral value, and comparing the integral value of the peak derived from the hydroxyl group-containing polymer. １ One preferred method for measuring the water content is to confirm the presence or absence of a water-soluble organic solvent by H-NMR, and if the solution contains only water as a solvent, to then measure the water content with a heat-drying moisture meter.

[0121] <Chemical Modification> Halogens can be introduced into the hydroxyl group-containing polymer by chemical modification, for example, by using a halide having a functional group (such as a carboxy group, an epoxy group, or an amino group) that reacts with a hydroxyl group.

[0122] <Cell Adhesion Factors and Cell-Interacting Proteins, etc.> The cell culture carrier may contain a cell adhesion factor and / or a cell-interacting protein, etc. By mixing the cell culture carrier with the cell adhesion factor and / or a cell-interacting protein, etc., a complex of the cell adhesion factor and / or the cell-interacting protein, etc. and the cell culture carrier can be obtained. By mixing, a halogen contained in the cell culture carrier binds to the cell adhesion factor and / or the cell-interacting protein, etc. through hydrophobic interaction, and / or a C═O group, typically or preferably contained in the hydroxyl group-containing polymer of the cell culture carrier, forms a covalent bond with the cell adhesion factor and / or the cell-interacting protein, etc., thereby forming a complex of the cell culture carrier and the cell adhesion factor and / or the cell-interacting protein, etc. Therefore, by simply and simply mixing, users can immobilize any cell adhesion factor and / or cell-interacting protein, etc., on the cell culture carrier of the present invention. Therefore, the present invention also encompasses cell culture carriers further comprising at least one selected from the group consisting of a cell adhesion factor, a cell-interacting protein, and a cell-interacting peptide present on the surface.

[0123] The immobilization method is not particularly limited, as long as a complex between the cell culture carrier and the cell adhesion factor and / or cell-interacting protein, etc. is formed. In one embodiment of the present invention, the cell culture carrier is mixed with a solution containing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells, and at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells can be immobilized on the surface of the cell culture carrier. For example, the cell adhesion factor and / or cell-interacting protein, etc. can be immobilized on the surface of the cell culture carrier by dissolving the cell adhesion factor and / or cell-interacting protein, etc. in PBS(-), mixing the resulting solution with a halogen-introduced hydroxyl group-containing polymer, and mixing at 120 rpm at room temperature for 16 hours or more.

[0124] From the viewpoint of being able to adjust the ratio of the cell adhesion factor and / or cell-interacting protein, etc. in the complex within a suitable range, the mass ratio of the cell culture carrier to the cell adhesion factor and / or cell-interacting protein, etc. to be mixed is preferably 1:0.0001 to 1:0.1, more preferably 1:0.001 to 1:0.01, and even more preferably 1:0.005 to 1:0.01. The masses of the cell culture carrier, cell adhesion factor, cell-interacting protein, etc. in this case are masses in a dry state.

[0125] <Optional Components Other Than Cell Adhesion Factors and Cell-Interacting Proteins, etc.> When a cell culture carrier contains an optional component (such as polymer microparticles) other than a cell adhesion factor and a cell-interacting protein, etc., a cell culture carrier containing the optional component can be prepared by mixing the optional component with a component other than the optional component. The mixing method is not particularly limited and may be selected appropriately. For example, the optional component may be mixed when mixing a hydroxyl group-containing polymer with a cell adhesion factor and / or cell-interacting protein, etc.; the optional component may be mixed with a complex in which the cell adhesion factor and / or cell-interacting protein, etc. is covalently bonded to a hydroxyl group-containing polymer and / or bound via hydrophobic interaction; or the optional component may be added when preparing an uncrosslinked polymer solution.

[0126] The cell culture carriers of the present invention have cell adhesive properties even without containing a cell adhesion factor derived from a living organism. The cell culture carriers of the present invention can also have any cell adhesion factor and / or any cell-interacting protein immobilized on their surface simply by mixing them with a cell adhesion factor. The cell culture carriers of the present invention on which cell adhesion factors and / or cell-interacting proteins are immobilized also have cell adhesive properties. Therefore, the cell culture carriers of the present invention can be suitably used in cell culture. The method for cell culture is not particularly limited, and known methods can be used.

[0127] <Culture Vessel> The culture vessel that can be used is not particularly limited, and commonly used culture vessels can be used. Examples include plates, well plates, flasks (including T-flasks, shaker flasks, spinner flasks, etc.), chamber slides, dishes, tubes, bottles, roller bottles, bags (including shaker bags), and bioreactors. Both homogeneous and heterogeneous bioreactors can be used. In homogeneous bioreactors, cells can freely contact the liquid medium and are dispersed throughout the reactor. Homogeneous bioreactors include stirred-bed, bubble-column, air-lift, and shaking bioreactors. In heterogeneous bioreactors, cells do not directly contact the liquid medium, but are stably attached or retained on the cell culture supports for stable culture. Examples of heterogeneous bioreactors include fixed-bed, fluidized-bed, stirred-bed, and hollow-fiber bioreactors. Here, a fixed-bed bioreactor is a bioreactor in which the cell culture supports are fixed and the liquid medium flows between the cell culture supports. Furthermore, a fluidized bed type bioreactor is one in which the cell culture carriers are not immobilized but are fluidized inside the reactor by the upward flow of the liquid medium. A stirred bed type bioreactor is one in which the cell culture carriers are fluidized by a stirring blade. The culture vessel can be freely selected depending on the purpose, from inoculation and seeding to production scale, but from the viewpoint of scale-up and adhering and growing cells on the cell culture carriers, the culture vessel is preferably a heterogeneous bioreactor. However, a homogeneous bioreactor may be used as the culture vessel in the present invention by placing cell culture carriers in it.

[0128] <Liquid medium> There are no particular limitations on the liquid medium that can be used, and any liquid medium suitable for the cells can be freely selected and used. Examples of liquid media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagle's Minimum Essential Medium; αMEM), and MEM medium (Minimum Essential Medium; αMEM). Examples of suitable liquid media include RPM11640 medium, Iscove's Modified Dulbecco's Medium (IMDM), and the like, and a liquid medium containing one or a mixture of two or more of these can be used. Of course, other media suitable for the cells to be cultured can also be used. The liquid medium may contain fetal bovine serum, or may be xeno-free without the addition of fetal bovine serum, or a completely synthetic medium that does not contain not only fetal bovine serum but also protein components and animal components may be used.

[0129] <Starting Cells> Starting cells that can be used are cells used as starting materials in the cell production method using the cell culture carrier of the present invention, and are cells that can grow on the surface of the cell culture carrier of the present invention. Examples of starting cells include pluripotent stem cells, tissue stem cells, somatic cells, mammalian cell lines used for the production of useful substances such as pharmaceuticals or for treatment, and insect cells.

[0130] Pluripotent stem cells are stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germ stem cells (GS cells).

[0131] Tissue stem cells refer to stem cells that are limited to certain tissues but have the ability to differentiate into various cell types (pluripotency). Examples include bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, liver stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, pancreatic stem cells (e.g., pancreatic duct epithelial stem cells), leukocyte stem cells, lymphocyte stem cells, and corneal stem cells.

[0132] Somatic cells refer to cells that make up multicellular organisms, and examples thereof include osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiac muscle cells, myoblasts, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fibrocytes, EB virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, hepatic parenchymal cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, and pancreatic β cells.

[0133] Examples of mammalian cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, and GP+ cells. E-86 cells, GP+envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA PaCG-2 cells, N18 cells, Namalwa cells, NG108-15 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER. C6 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2 / O-Ag14 cells, ST2 cells, THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells, ψ2 cells, and ψCRE cells.

[0134] Examples of insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, Anemone cells, Anemone cells, Spodoptera frugiperda cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila cells, Boettcheris caudatus cells, Aedes albopictus cells, swallowtail butterfly cells, American cockroach cells, and nettle looper cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.).

[0135] These cells may be aggregated together or may be differentiated. The aggregated cells may have the function of an organ. The cells may be immediately collected from a living body or may be cultured. The cells collected from a living body may have formed an organ.

[0136] <Optional Components> Examples of optional components that may be contained in the composition contained in the culture vessel include serum substitutes, cell growth factors / differentiation factors, cytokines, hormones, polyanions, polyvinyl alcohol, and transferrin. These optional components can be used alone or in combination of two or more. Examples of serum substitutes include insulin, growth factors / differentiation factors, transferrin, and proteins such as albumin.

[0137] Examples of cell growth factors and differentiation factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), osteonectin, angiopoietin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), Examples of such proteins include nerve growth factor (NGF), leukemia inhibitory factor (LIF), stem cell factor (SCF), bone morphogenetic protein (BMP), interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), Notch ligand (Delta-1, Delta-3, Delta-4, Jagged-1, Jagged-2, etc.

[0138] Examples of cytokines include interleukin-1α, interleukin-1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interferon α, interferon β, interferon γ, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), MCP-1, erythropoietin (EPO), thrombopoietin (TPO), and Flk-2 / Flt-3 ligand (FL).

[0139] Examples of hormones include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadothrombin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, leptin, luteinizing hormone, melanocyte Examples of hormones that may be involved in the production of steroid hormones include steroid hormones, such as steroid hormone, steroid hormone, steroid hormone-releasing hormone, steroid hormone-stimulating hormone, steroid hormone-releasing ...

[0140] Examples of polyanions include heparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate, etc. These polyanions stabilize the cell growth factors and differentiation factors by binding to them, thereby promoting cell proliferation more efficiently.

[0141] Polyvinyl alcohol (PVA) can be freely selected from the vinyl alcohol polymers and modified PVA. When culturing cells, bovine serum albumin (BSA), human recombinant albumin, etc. are often added to the culture medium for the purposes of cell protection, growth factor stabilization, etc. By using PVA, a serum-free culture environment can be created in which proteins such as BSA are excluded. The content of PVA in the serum-free medium is preferably 0.01 to 0.5% by mass, more preferably 0.05 to 0.2% by mass.

[0142] <Culturing step> The conditions for the culturing step may be appropriately selected from commonly used conditions depending on the type of raw material cells or culture vessel. For example, the conditions are: temperature 37°C, humidity 95%, CO ２ It can be cultured under conditions of a CO concentration of 5%. ２ The concentration may be 1 to 10%, or 3 to 8%.

[0143] <Detachment step> The cultured cells can be detached from the cell culture carriers and collected. The detachment method is not particularly limited, and can be carried out by a known method. Such methods include a method using a chelating agent, a method using a protease, a method of detachment by physical force, and a combination thereof.

[0144] <Separation step> Next, the detached cells are separated from the cell culture carriers, allowing the produced cells and the cell culture carriers from which the cells have been detached to be recovered. The separation method is not particularly limited, but for example, separation by filtration and separation utilizing the difference in sedimentation velocity between the detached cells and the cell culture carriers can be employed. In separation utilizing the difference in sedimentation velocity, if the culture vessel is left standing for a certain period of time after the detachment step, the cell culture carriers will settle to the bottom of the culture vessel, and the cells will be suspended in the culture vessel. Utilizing this, the liquid containing the cells can be recovered by sucking the liquid from the liquid surface in the culture vessel, and the liquid can be separated from the cell culture carriers.

[0145] Optionally, the separated cell culture carriers can be washed to recover cells that were attached to the surface of the cell culture carriers or present near the cell culture carriers. For example, the cells can be recovered by mixing the separated cell culture carriers with a washing solution such as water or a buffer solution, and then performing the above-mentioned separation step again. The amount of washing solution, the mixing method or conditions, etc. are not particularly limited and may be selected appropriately. Furthermore, when this washing step is performed, there is no limit to the number of times, and it may be performed once or multiple times.

[0146] Thereafter, the liquid containing the cells can be subjected to a conventionally known method, such as centrifugation or the use of a cell fractionation filter, to recover the cells produced by the cell culture method using the cell culture carrier of the present invention.

[0147] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Physical properties in the examples and comparative examples were measured according to the following procedures.

[0148] <Saturated PBS(-) Content> The hydroxyl group-containing polymer (hydrogel particles) was dried under reduced pressure at room temperature until it reached a constant weight. 0.2 g of the dried hydrogel particles was weighed out, and a sufficient amount of PBS(-) was added to cause swelling. The swollen hydrogel particles were all recovered by filtration, and the PBS(-) adhering to the surface was wiped off, after which the mass was measured. From the difference in mass before and after swelling, the saturated PBS(-) content of the hydroxyl group-containing polymer was calculated using the following formula. Weighing and swelling were performed at a temperature of 25°C. Saturated PBS(-) Content [mass %] = [{(mass of swollen hydrogel particles [g]) - (mass of dry hydrogel particles [g])} / (mass of swollen hydrogel particles [g])] x 100

[0149] <Average particle size> Dry hydrogel particles (approximately 20 mg) were mixed with PBS(-) (approximately 5 mL) and allowed to swell at room temperature for 2 hours to prepare a mixed solution. The swollen particles in PBS(-) were photographed under a microscope, and the distance between two points on 50 randomly selected particles on the image was measured to determine the particle diameter, and the average of these measurements was taken as the average particle size.

[0150] <X-ray photoelectron spectroscopy (XPS)> Cell culture carriers dried under reduced pressure at room temperature until they reached a constant weight were subjected to XPS measurement under the following conditions: Instrument type: PHI Quantera SXM (scanning X-ray photoelectron spectroscopy) X-ray source: monochromated Al Kα (1486.6 eV) X-ray beam diameter: 100 μmφ (25 W, 15 kV) Measurement range: 1000 μm (horizontal) × 300 μm (vertical) Signal acquisition angle: 45° Charge neutralization conditions: neutralization electron gun, Ar+ ion gun Vacuum degree: 1 × 10 －６Pa Measurement method: Normal measurement, no sample washing. Next, the halogen content in the cell culture carrier was determined from the surface element composition ratio, and the molar ratio of C=O to C-C in the hydroxyl group-containing polymer was determined from the peak area in the C1s spectrum. In addition, the ratio of the OH peak area to the area in the O1s spectrum was determined.

[0151] Example 1 Synthesis of a vinyl alcohol polymer having an ethylenically unsaturated group 40 g (monomer repeating unit: 908 mmol) of raw material PVA (trade name "PVA11", degree of polymerization: 1700, degree of saponification: approximately 98.0 to 99.0 mol%, viscosity (4%, 20°C): 25.0 to 31.0 mPa·s, manufactured by Kuraray Co., Ltd.) was placed in a separable flask equipped with a 1 L Dimroth condenser, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw material PVA had dissolved, 2.1 g (18.7 mmol) of vinyl methacrylate was added while heating and stirring at 80°C, and stirring was continued for an additional 3 hours at 80°C. After allowing to cool, the reaction solution was poured into 2 L of methanol while stirring. Stirring was stopped and the mixture was left as is for 1 hour. The resulting solid was collected and then further washed by immersing in 1 L of methanol for 1 hour. This washing procedure was repeated a total of three times. The collected solid was dried in vacuum at room temperature overnight to obtain methacryloylated PVA117. The introduction rate of ethylenically unsaturated groups (methacryloyl groups) in this methacryloylated PVA117 was 2.0 mol% relative to the repeating units of the raw material PVA (hereinafter referred to as "MA-PVA117(2.0)").

[0152] <Preparation of Dried Hydrogel Particles> 440 mL of ion-exchanged water was added to 60 g of MA-PVA117(2.0) and dissolved with stirring at 80°C for 4 hours. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to and dissolved in this MA-PVA117(2.0) aqueous solution to a concentration of 0.1% by mass to prepare an uncrosslinked polymer solution. 3300 mL of liquid paraffin and 8 g of Span 80 were placed in a separable flask equipped with a 5 L Dimroth condenser, and the uncrosslinked polymer solution was slowly added to the flask. This mixture was stirred at 350 rpm with a mechanical stirrer to form a W / O dispersion, which was then heated to 40°C in a water bath and purged with nitrogen for 30 minutes. Stirring was then continued at 70°C for 3 hours. After cooling to room temperature, the liquid paraffin containing the dispersed hydrogel particles was filtered through a 100 μm mesh. The obtained hydrogel particles were washed with a total of 3 L of hexane to remove the liquid paraffin, and the obtained hydrogel particles were classified using a JIS standard sieve to have a particle size of 180 to 300 μm. The hydrogel particles were then further dehydrated by adding them to 1 L of acetone, and dried under reduced pressure to obtain dried hydrogel particles (hereinafter referred to as "MA-PVA117(2.0) gel particles").

[0153] <Plasma Treatment> 10 g of MA-PVA117(2.0) gel particles were placed in a quartz bottle (volume 1 L). While the quartz bottle was rotated around its longitudinal axis between two electrodes and carbon tetrafluoride gas was passed through the quartz bottle at a rate of 150 cc / min, a voltage was applied under the conditions shown in Table 1 below, yielding dried fluorine-containing hydrogel particles. The average particle size of the hydrogel particles in an equilibrium swollen state in PBS(-) was 272 μm.

[0154] <Evaluation of cell adhesion - shaking culture> Cultivation area: 75 cm ２ The cell culture carriers thus prepared were placed in a cell culture flask with a surface area of ​​162 cm. ２ 30 mL of 10% calf serum-supplemented D-MEM (low glucose) was added thereto, and then approximately 8.1 × 10 NIH / 3T3 cells were added. ５ cells, and CO ２The cells were shaken at 85 rpm in an incubator at 37° C. for 4 days. Subsequently, the cells were detached from the cell culture carriers using a 0.25% trypsin-EDTA solution, and the number of recovered cells was measured using a disposable cell counting chamber.

[0155] Examples 2 to 4 and 7 to 8 Cell culture carriers were obtained and evaluated in the same manner as in Example 1, except that the plasma treatment conditions were changed as shown in Table 1.

[0156] The cell culture carriers produced in Examples 3 and 4 were also evaluated for cell adhesion by static culture according to the following procedure. <Evaluation of cell adhesion - static culture> 5 mg of the produced cell culture carriers was weighed out and placed in a 24-well plate. 1 mL of 10% calf serum-supplemented D-MEM (low glucose) was added thereto, and then NIH / 3T3 cells were cultured at approximately 1.5 x 10 per well. ４ cells, and CO ２ The plates were placed in an incubator at 37°C for 4 days. Subsequently, 1 mL of reagent (CellTiter-Glo® 2.0 Cell Viability Assay) was added per well, and the plates were shaken for 30 seconds. After that, the plates were placed at room temperature for 10 minutes in the dark, and 100 μL of the supernatant was collected into a 96-well plate. The luminescence intensity was measured using a microplate reader and used as an index of cell activity.

[0157] Comparative Example 1 Cell culture carriers were obtained and evaluated in the same manner as in Example 1, except that oxygen gas was used instead of carbon tetrafluoride gas during the plasma treatment.

[0158] Comparative Example 2 Cell culture carriers were obtained and evaluated in the same manner as in Example 1, except that ammonia gas was used instead of carbon tetrafluoride gas during the plasma treatment.

[0159] Comparative Example 3 Cell culture carriers were obtained and evaluated in the same manner as in Comparative Example 2, except that the conditions for the plasma treatment were changed.

[0160] Comparative Example 4: Dried hydrogel particles were prepared in the same manner as in Example 1, and collagen was then immobilized on untreated hydrogel particles that had not been plasma-treated. Specifically, porcine atelocollagen (Nitta Gelatin Co., Ltd.) was first dissolved in PBS(-) to a concentration of 200 mg / L to prepare a collagen solution. 1 g of untreated hydrogel was added to 30 mL of this collagen solution and stirred overnight at room temperature. After stirring, the cell culture carrier was washed four times with 30 mL of ion-exchanged water, then substituted three times with 20 mL of ethanol, and then vacuum-dried overnight at room temperature to obtain a sample. After the sample was saturated with PBS(-), the amount of collagen immobilized in the sample was measured using a Collagen Quantitation Kit (Cosmo Bio Co., Ltd.). The value was below the detection limit, indicating that collagen was not immobilized.

[0161] Example 5 Collagen was immobilized on the cell culture carriers produced in Example 3. Specifically, porcine atelocollagen (Nitta Gelatin Co., Ltd.) was first dissolved in PBS(-) to a concentration of 200 mg / L to prepare a collagen solution. 1 g of the cell culture carriers produced in Example 3 was added to 30 mL of this collagen solution and stirred overnight at room temperature. After stirring, the cell culture carriers were washed four times with 30 mL of ion-exchanged water, then substituted three times with 20 mL of ethanol, and then vacuum-dried overnight at room temperature to obtain collagen-immobilized cell culture carriers. The dried cell culture carriers were swelled to saturation with PBS(-), and the amount of collagen immobilized in the collagen-immobilized cell culture carriers was measured using a Collagen Quantitation Kit (Cosmo Bio Co., Ltd.). The result was 10 μg / wet-100 mg. Furthermore, the cell adhesiveness of the collagen-immobilized cell culture carriers was evaluated in the same manner as in Example 3, and good cell adhesiveness was confirmed in both static and shaking culture.

[0162] Example 6 Proteins were immobilized on the cell culture carriers prepared in Example 1. Specifically, 1 mL of 500 mg / L iMatrix-511 (MATRIXOME) was mixed with 3.6 mL of PBS(-), and 0.4 g of the cell culture carriers prepared in Example 1 that had been saturated and swollen with PBS(-) was added and stirred overnight at room temperature. After stirring, the cell culture carriers were washed three times with 5 mL of PBS(-), yielding cell culture carriers with immobilized iMatrix-511. The amount of protein immobilized on the cell culture carriers was measured using a BCA Protein Assay Kit (Takara Bio Inc.), yielding a value of 78 μg / wet-100 mg. Furthermore, the cell adhesiveness of the cell culture carriers with immobilized proteins was evaluated in the same manner as in Example 3, and favorable cell adhesiveness was confirmed in both static and shaking culture.

[0163] Example 9 Proteins were immobilized on the cell culture carriers prepared in Example 1. Specifically, 0.3 mL of 500 mg / L Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) was mixed with 2.7 mL of PBS(-), and 0.1 g of the cell culture carriers prepared in Example 1 was added and stirred at room temperature for 1 hour. After stirring, the cell culture carriers were washed three times with 5 mL of PBS(-) to obtain cell culture carriers with VTN-N immobilized. The amount of protein immobilized on the protein-immobilized cell culture carriers was measured using a BCA Protein Assay Kit (Takara Bio Inc.), and the results are shown in Table 2. Furthermore, the cell adhesiveness of the protein-immobilized cell culture carriers was evaluated using adipose-derived MSCs in the same manner as in Example 3. The cell adhesiveness was confirmed in shaking culture as shown in Table 2.

[0164] Examples 10 to 13 Cell culture carriers were obtained and evaluated in the same manner as in Example 9, except that the plasma treatment conditions were changed as shown in Table 2.

[0165]

[0166]

[0167] The cell culture carriers of the present invention have cell adhesive properties even without containing biologically derived cell adhesion factors. Therefore, users can use the cell culture carriers of the present invention without worrying about the safety associated with biologically derived cell adhesion factors. Furthermore, the cell culture carriers of the present invention can immobilize cell adhesion factors on their surfaces simply by mixing them with the cell adhesion factors. Therefore, users can easily and simply immobilize any desired cell adhesion factors on the cell culture carriers of the present invention, and the cell culture carriers thus obtained can be used for cell culture. Furthermore, the cell culture carriers of the present invention can immobilize proteins, peptides, etc. that interact with cells on their surfaces simply by mixing them with the proteins, peptides, etc. Therefore, users can easily and simply immobilize any desired cell-interacting proteins, etc. on the cell culture carriers of the present invention.

Claims

1. A cell culture carrier comprising a polymer having a hydroxyl group and a halogen, wherein the halogen content of the cell culture carrier measured by X-ray photoelectron spectroscopy is 3 to 60 atomic %, and the saturation content of the polymer in phosphate buffered saline (PBS(-)) at 25°C is 10 to 99 mass %.

2. The cell culture carrier according to claim 1, wherein the polymer is a hydrogel-forming polymer.

3. The cell culture carrier according to claim 2, wherein the hydrogel-forming polymer in an equilibrium swollen state in phosphate buffered saline (PBS(-)) is in the form of particles with an average particle size of 10 to 5000 μm.

4. The cell culture carrier according to any one of claims 1 to 3, wherein the ratio of the OH peak area to the O1s spectrum area measured by X-ray photoelectron spectroscopy of the cell culture carrier is 1 to 100%.

5. The cell culture carrier according to any one of claims 1 to 4, wherein the halogen is introduced by plasma treatment.

6. The cell culture carrier according to any one of claims 1 to 5, wherein the halogen is fluorine.

7. The cell culture carrier according to any one of claims 1 to 6, wherein the polymer has one or more shapes selected from the group consisting of irregular particles, spherical particles, micromolded bodies, arbitrarily shaped articles formed by a 3D printer, films, threads, fabrics, hollow fibers, porous monoliths, and coated articles.

8. The cell culture carrier according to any one of claims 1 to 7, wherein the molar ratio of C=O to C-C of the cell culture carrier is 8 to 90 as measured by X-ray photoelectron spectroscopy.

9. The cell culture carrier according to any one of claims 1 to 8, further comprising at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells present on the surface.

10. The cell culture carrier according to claim 9, wherein the cell adhesion factor is a peptide.

11. A method for producing a cell culture carrier, comprising mixing the cell culture carrier according to any one of claims 1 to 8 with a solution containing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells, and immobilizing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells on the surface of the cell culture carrier.

12. The method for producing a cell culture carrier according to claim 11, wherein the cell adhesion factor is a peptide.

13. A method for producing a cell culture carrier containing a polymer having hydroxyl groups and a halogen, the method comprising subjecting the polymer having hydroxyl groups to plasma treatment in the presence of a halogenated hydrocarbon gas, wherein the halogen content of the cell culture carrier measured by X-ray photoelectron spectroscopy is 3 to 60 atomic %, and the saturation content of the polymer in phosphate buffered saline (PBS(-)) at 25°C is 10 to 99 mass %.

14. The method of claim 13, wherein the polymer is a hydrogel-forming polymer.

15. The method of claim 13 or 14, wherein the polymer in the dry state is subjected to a plasma treatment.

16. The method according to any one of claims 13 to 15, wherein the plasma treatment is a treatment in which plasma is irradiated at an irradiation intensity of 50 W or more.

17. The method according to any one of claims 13 to 16, wherein the obtained cell culture carrier is mixed with a solution containing at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells, and at least one selected from the group consisting of a cell adhesion factor, a protein that interacts with cells, and a peptide that interacts with cells is further immobilized on the surface of the cell culture carrier.

18. The method of claim 17, wherein the cell adhesion factor is a peptide.

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